Compositions and methods for treating cancer

ABSTRACT

The invention provides compositions and methods for treating ovarian cancer. Specifically, the invention relates to administering a genetically modified T cell having α-folate receptor (FRα) binding domain and 4-1BB (CD137) costimulatory domain to treat ovarian cancer.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation application of, and claimspriority to, U.S. patent application Ser. No. 16/594,871, filed Oct. 7,2019, (allowed), a continuation of U.S. patent application Ser. No.15/212,916, filed Jul. 18, 2016, issued as U.S. Pat. No. 10,457,729,which is a continuation of U.S. patent application Ser. No. 13/979,927,filed Nov. 1, 2013, issued as U.S. Pat. No. 9,402,865, a U.S. nationalphase application filed under 35 U.S.C. § 371 claiming benefit toInternational Patent Application No. PCT/US2012/021738, filed on Jan.18, 2012, which is entitled to priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 61/433,731, filed on Jan. 18, 2011,each of which application is hereby incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Ovarian cancer is responsible for the majority of gynecologic cancerdeaths. In 2004, in the United States, 25,580 new cases were diagnosedand 16,090 women died of ovarian cancer.

The disease is more common in industrialized nations, with the exceptionof Japan. In the United States, females have a 1.4% to 2.5% (1 out of40-60 women) lifetime chance of developing ovarian cancer. Older womenare at highest risk.

Although intraperitoneal chemotherapy has been recommended as a standardof care for the first-line treatment of ovarian cancer, the basis forthis recommendation has been challenged. Radiation therapy is noteffective for advanced stages because when vital organs are in theradiation field, a high dose cannot be safely delivered. Surgicaltherapy is also not also effective.

Despite the initial successful multimodality therapy with cytoreductivesurgery and subsequent combination chemotherapy, most patients withadvanced disease will ultimately relapse and become incurable. For thisreason, novel therapeutic approaches for the treatment of thismalignancy are urgently needed.

Ovarian cancer in particular appears to be suited to adoptive transferapproach based on the fact that the ovarian tumors are relativelyimmunogenic, inducing an endogenous T cell response.

Accordingly, there exists a need for improved therapeutic modalities toprovide anti-tumor immunity, and thereby treat ovarian and othercancers.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the isolated nucleicacid sequence comprises the nucleic acid sequence of α-folate receptor(FRα) binding domain and the nucleic acid sequence of 4-1BB (CD137)costimulatory domain.

In one embodiment, the nucleic acid sequence further comprises thenucleic acid sequence of CD3 zeta binding domain.

In one embodiment, the CAR comprises the amino acid sequence of SEQ IDNO: 13 or SEQ ID NO: 22.

In one embodiment, the isolated nucleic acid sequence encoding the CARcomprises the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 20.

In one embodiment the FRα binding domain is an antibody or a FRα-bindingfragment thereof. Preferably, the FRα binding domain is a Fab or a scFV.

In one embodiment, the FRα binding domain binds to a tumor antigen,wherein the tumor antigen is FRα. In one embodiment, the tumor antigenis associated with an epithelial malignancy. In another embodiment, thetumor antigen is associated with a solid tumor.

In one embodiment, the FRα binding domain comprises the amino acidsequence of SEQ ID NO: 15 or SEQ ID NO: 23.

In one embodiment, the FRα binding domain is encoded by the nucleic acidsequence of SEQ ID NO: 3 or SEQ ID NO: 21.

In one embodiment, the 4-1BB costimulatory domain comprises the aminoacid sequence of SEQ ID NO: 18.

In one embodiment, the 4-1BB costimulatory domain is encoded by thenucleic acid sequence of SEQ ID NO: 6.

In one embodiment, the CD3 zeta signaling domain comprises the aminoacid sequence of SEQ ID NO: 19.

In one embodiment, the CD3 zeta signaling domain is encoded by thenucleic acid sequence of SEQ ID NO: 7.

In one embodiment, the isolated nucleic acid sequence further comprisesthe nucleic acid sequence of a transmembrane domain.

The invention also provides an isolated CAR comprising a FRα bindingdomain and a 4-1BB costimulatory domain.

The invention also provides a genetically modified T cell comprising anisolated nucleic acid sequence encoding a CAR, wherein the isolatednucleic acid sequence comprises the nucleic acid sequence of a FRαbinding domain and the nucleic acid sequence of a 4-1BB costimulatorydomain.

The invention also provides a vector comprising an isolated nucleic acidsequence encoding a CAR, wherein the isolated nucleic acid sequencecomprises the nucleic acid sequence of a FRα binding domain and thenucleic acid sequence of a 4-1BB costimulatory domain.

The invention also provides a method for providing anti-tumor immunityin a subject. In one embodiment comprises administering to the subjectan effective amount of a genetically modified T cell comprising anisolated nucleic acid sequence encoding a CAR, wherein the isolatednucleic acid sequence comprises the nucleic acid sequence of a FRαbinding domain and the nucleic acid sequence of a 4-1BB costimulatorydomain, thereby providing anti-tumor immunity in the subject. In oneembodiment, the isolated nucleic acid sequence further comprises thenucleic acid sequence of a CD3 zeta signaling domain.

In one embodiment, the presence of the costimulatory domain enhances Tcell survival. In another embodiment, the presence of the costimulatorydomain increases the efficacy of anti-tumor immunity in a subject.

In one embodiment, the subject is a mammal. Preferably, the subject is ahuman.

The invention also provides a method for stimulating a T cell-mediatedimmune response to a cell population or tissue in a subject. In oneembodiment, the method comprises administering to the subject aneffective amount of a genetically modified T cell comprising an isolatednucleic acid sequence encoding a CAR, wherein the isolated nucleic acidsequence comprises the nucleic acid sequence of a FRα binding domain andthe nucleic acid sequence of a 4-1BB costimulatory domain, therebystimulating a T cell-mediated immune response in a subject.

The invention also provides a method for treating ovarian cancer in asubject. In one embodiment, the method comprises administering to thesubject an effective amount of a genetically modified T cell comprisingan isolated nucleic acid sequence encoding a CAR, wherein the isolatednucleic acid sequence comprises the nucleic acid sequence of a FRαbinding domain and the nucleic acid sequence of a 4-1BB costimulatorydomain, thereby treating the ovarian cancer in the subject.

The invention also provides a method for treating cancer in a subject.In one embodiment, the method comprises administering to the subject aneffective amount of a genetically modified T cell comprising an isolatednucleic acid sequence encoding a CAR, wherein the isolated nucleic acidsequence comprises the nucleic acid sequence of a FRα binding domain andthe nucleic acid sequence of a 4-1BB costimulatory domain, therebytreating cancer in the subject.

The invention also provides a method of generating a persistingpopulation of genetically engineered T cells in a subject diagnosed withovarian cancer. In one embodiment, the method comprises administering tothe subject an effective amount of a genetically modified T cellcomprising an isolated nucleic acid sequence encoding a CAR, wherein theisolated nucleic acid sequence comprises the nucleic acid sequence of aFRα binding domain and the nucleic acid sequence of a 4-1BBcostimulatory domain, wherein the persisting population of geneticallyengineered T cells persists in the subject for at least one month afteradministration. In one embodiment, the persisting population ofgenetically engineered T cells persists for at least three months afteradministration.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1B are a set of images showing the construction and lentiviralgene transfer of αFR CARs to human T cells.

FIGS. 2A-2C are a series of graphs demonstrating that CAR+ T cellspreferentially secreted Th1 cytokines.

FIGS. 3A-3B are a set of graphs showing direct and specific tumorrecognition and killing of αFR+ human ovarian cancer.

FIGS. 4A-4C are set of images and a graph showing that incorporation ofthe 4-1BB signaling domain can enhance anti-tumor activity in Winnassay.

FIGS. 5A-5E, are a series of images showing treatment of large,established human ovarian cancer using CAR gene therapy: 4-1BBcostimulation mediates enhanced T cell survival. FIG. 5A shows arepresentative experimental model. FIG. 5B is a chart depicting tumorvolume. FIG. 5C is a series of images depicting tumor growth. FIG. 5D isa chart depicting tumor volume. FIG. 5E is a chart depicting cell countsfor CD4 and CD8 T cells.

FIG. 6 is an image representing chimeric anti-alpha folate receptorimmunoreceptor α-FR 4-1BB:CD3ζ (transgene and vector construct. Theconstruct was cloned into the pELNS backbone vector (bottom), whichcontains the packaging signal (ψ), the central polypurine tract/centraltermination sequence (cppt/CTS) and the elongation factor 1-α promoter(ef-1α). The transfer vector is driven off of the 5′ LTR duringpackaging and the 3′ SIN LTR is copied to the 5′ end upon reversetranscription.

FIG. 7 is an image showing the plasmid map of PELNS—MOv19-4-1BB-CD3zeta.

FIGS. 8A-8B are a series of images showing the generation and cytolyticactivity of anti-αFR lentiviral vector-engineered T cells. FIG. 8A showsa schematic representation of the αFR-binding chimeric receptors. Abinding-control chimeric receptor with a truncated TCRζ domain and aspecificity control receptor with an anti-CD19 scFv were alsoconstructed. FIG. 8B is an image showing expression of the αFR-CARproteins was examined on human primary CD4 T cells. Transductionefficiencies are determined by flow cytometry.

FIG. 9A is a series of images showing that the cell surface expressionof FR on AE17, AE17.FR, SKOV3 was determined by flow cytometry. Cellswere incubated with either the mouse anti-human αFR antibody MOV18(light gray histograms) or an isotype control (dark gray histograms)followed by staining with a FITC-conjugated goat anti-mouse Ig. FIG. 9Bis a series of plots showing cytolytic activity of the chimericreceptors on primary human T cells targeting cell lines expressing αFRdetermined using a 4 h ⁵¹Cr release assay. FRα+ tumor targets candirectly induce T cell cytokine secretion. FIG. 9C is a plot showingresults expressed as a mean and SD of triplicate wells from 1 of atleast 3 separate experiments.

FIG. 10 is a series of images showing efficient αFR-specific killing ofAE17.FR tumor cells in vitro. AE17 and AE17.FR cells transduced with GFPincubated CAR+ T cells for about 20 h at the indicated ratios, afterwhich cells were photographed under fluorescence microscopy. The CARtransduction efficiency for each group of T cells was ˜40%-50%.

FIGS. 11A-11B are a series of images showing anti-tumor activity of αFRchimeric receptor transduced T cells in vivo. NOD/scid/IL2rγ^(−/−) (NOG)mice were injected s.c. of SKOV3 Luc(1×10⁶ cells/mouse) mixed with CARexpressing T cells (1×10⁶ cells/mouse). Mixing of cells was performedimmediately before injection to minimize T cell and target interaction.The animals were imaged after inoculation every 10 days to evaluatetumor growth, and photon emission from luciferase-expressing cells wasquantified using the “Living Image” software.

FIGS. 12A-12D are a series of images showing that αFR retargeted T cellseradicate large pre-established tumors in vivo: effect of costimulatorysignaling domains and route of administration. FIGS. 12A and 12Cdemonstrate that mice subcutaneously injected with 3×10⁶ SKOV3 cellswere monitored for tumor growth until reaching tumor volume of 200˜300mm³. Tumor-bearing mice were treated with intratumoral injections of20×10⁶ T cells (˜40%-50% transgene positive) on day 40 and 45. FIGS. 12Band 12D demonstrate that SKOV3-bearing NOG mice were treated with Tlymphocytes expressing the BBz chimeric receptors via IT, IP, and IVroutes and the effect on tumor growth was assessed.

FIG. 13 is a graph showing that 4-1BB signals enhance the persistence ofhuman T lymphocytes in vivo. Peripheral blood was obtained fromretro-orbital bleeding on 20 day 74 and stained for the presence ofhuman CD45, CD4, and CD8 T cells. After gating on the human CD45+population, the CD4+ and CD8+ subsets were quantified using TruCounttubes (BD Biosciences). Persistence was greatest in the BBz groupindependent of route of injection.

FIGS. 14A-14C are a series of graphs and images showing that αFR CAR BBzeradication of SKOV3 tumor is antigen-specific. FIGS. 14A and 14B showSKOV3 bearing mice treated with lymphocytes expressing the BBz CAR(against αFR or CD19) and GFP on day 40 and 45. FIG. 14C showsperipheral blood from SKOV3-bearing NOG mice was obtained 3 weeks aftersecond time T cells injection and quantified for the presence of CD4 andCD8 T cells by a FACS Trucount assay.

FIGS. 15A-15C are a series of images showing that αFR CAR BBz specific Tcells inhibit tumor growth and ascites formation in SKOV3 murine modelof peritoneal carcinomatosis. FIG. 15A is an image showing i.p.injection of SKOV3 tumors in NOG mice results in abdominal distensionand nodular peritoneal tumors following CD19 CAR T cells treatment. Micedeveloped ascites as evidenced by a distended abdomen (middle) whencompared with a mouse (left) treated with FR CAR BBz T cells, postmortemvisualization of the peritoneum shows nodular tumor masses (arrows)within the abdominal cavity (right). FIGS. 15B and 15C showi.p./i.v.injection of αFR CAR BBz T cells delays tumor progression andascites formation, and improves survival. Kaplan-Meier survival curve ofNOG mice treated with either CD19 CAR or αFR CART cells.

FIG. 16 is a series of images showing that the adoptive transfer of αFRCAR BBz-specific T cells induces regression of ovarian cancer lungmetastasis. While tumors regressed in response to injection ofαFR-specific T cells, tumors grew progressively in CD19-specific T cellstreated mice.

FIG. 17 is a graph showing that CD4 T cells isolated from a healthydonor were transduced at an MOI of 20 with a GFP-expressing HIV derivedlentiviral vector, and cultured for 29 days. The X-axis representsfold-expansion (circles) or percent GFP expression (triangles).Transduced cells are open symbols and mock transduced cells are closedsymbols.

FIGS. 18A-18E are a series of plots and images depicting the generationand specific immune recognition by FRα CAR-transduced human T cells invitro. FIG. 18A shows schematic representation of MOv19-based CARconstructs containing the CD3 (cytosolic domain alone (MOv19-ζ) or incombination with CD137 costimulatory module (MOv19-BBζ). FRα-specificCAR with a truncated CD3ζ (domain (MOv19-Δζ) and anti-CD19-BBζ CAR areshown. VL, variable L chain; L, linker; VH, variable H chain; TM,transmembrane region. FIG. 18B depicts MOv19 CAR expression (solid blackline) on human CD3-gated cells after transduction with lentiviruscompared with parallel untransduced T cells (filled gray histograms).Percent transduction is indicated. FIG. 18C depicts surface FRαexpression (solid black line) by various human ovarian cancer cell linesby flow cytometry; isotype antibody control (filled gray histograms).FIG. 18D depicts antigen-specific IFN-γ secretion by MOv19-ζ andMOv19-BBζ CAR-transduced T cells but not MOv19-Δζ anti-CD19-BBζ T cells,following overnight incubation with FRα⁺ cancer cell lines. Mean IFN-γconcentration±SEM (pg/mL) from triplicate cultures is shown. FIG. 18Edepicts antigen-specific killing of FRα⁺ tumor cells by FRα CAR⁺ CD8+ Tcells in 18-hour bioluminescence assay at the indicated E/T ratio.Untransduced T cells (UNT) or gfp-transduced human CD8⁺ T cells servedas controls.

FIGS. 19A-19D are a series of graphs and images that show humanMOv19-BBζ CAR T cells eradicate large pre-established tumors in vivo,and showing the effect of CD137 costimulatory signaling domains androute of administration. NSG mice bearing established s.c. tumor weretreated with intratumoral injections of 8×10⁶ CAR⁺ T cells on days 0 and5 and imaged every 2 weeks. FIG. 19A depicts tumor growth, as assessedby caliper measurement [V=½(length×width²)]. FIG. 19B shows that SKOV3fLuc⁺ bioluminescence signal was decreased in MOv19-BBζ CAR treated micecompared with the MOv19-ζ and the control treatment groups 2 weeks and 4weeks after last T-cell dose. SKOV3 fLuc-bearing NSG mice were treatedwith 8×10⁶ MOv19-BBζ T cells via i.t., i.p., or i.v. routes. FIG. 19Cdepicts tumor growth, as assessed by caliper measurement. FIG. 19D showsthat CD137 signaling enhances the survival of human CD4⁺ and CD8⁺ Tcells in vivo on day 73 (4 weeks following last T-cell dose) in theperipheral blood. CD4 and CD8 T cells were quantitated from blood byusing the TruCount method. Mean cell concentration (cells/μL)±SD for allevaluable mice in each treatment group is shown.

FIGS. 20A-20D are a series of graphs showing that tumor eradication byCAR T cells is antigen-specific. NSG mice with s.c. SKOV3 fLuc⁺ tumorwere treated with 8×10⁶ T cells (40% transduction efficiency) expressingMOv19-BBζ, anti-CD19-BBζ, or gfp via i.t. infusion on days 0 and 5. FIG.20A depicts measurements of tumor volume by calipers every 2 to 3 days.Peripheral blood was collected 3 weeks following last T-cell infusion.FIG. 20B depicts the absolute number of human CD4+ and CD8+ T cells/μlof blood. Mean cell count±SD is shown. FIG. 20C depicts FRα- andCD19-specific CAR expression on human CD3+ T cells from peripheral bloodof treated mice measured by flow cytometry by using goat anti-mouse IgGF(ab′)2. Mean CAR⁺ expression frequency±SD per group is shown. FIG. 20Ddepicts absolute CAR+ T-cell count, calculated as number of human CD3+ Tcells/L of blood multiplied by percent CAR+. Mean count±SD wasdetermined.

FIG. 21 is a series of images demonstrating that CAR T-cell localizationto tumor in vivo is antigen-specific. NSG mice with s.c. SKOV3 fLuc⁺tumors were treated with i.v. injections of 8×10⁶ T cells expressingMOv19-BBζ (top), anti-CD19-BBζ (middle), or gfp (bottom) on days 0 and5. SKOV3 tumors grown for approximately 40 additional days werecollected from euthanized mice and stained for human CD3 expression(brown). Representative sections are shown at ×100 magnifications.

FIGS. 22A-22D are a series of images and a plots showing that Mov19-BBζT cells inhibit tumor growth and ascites formation in SKOV3 murine modelof peritoneal carcinomatosis. FIG. 22A depicts NSG mice which receivedi.p. injection of 5×10⁶ SKOV3 fLuc⁺ tumor cells and were randomized into4 groups before beginning therapy with 9×10⁶ T cells expressingMOv19-BBζ or anti-CD19-BBC via i.p. or i.v. infusion on day 30 and 35after tumor inoculation. FIG. 22B depicts representative NSG micetreated with MOv19-BBζ T cells (left) via i.v. (top) or i.p (bottom)infusion. Mice treated with anti-CD19-BBζ T cells (right) developedascites as evidenced by a distended abdomen (middle). Postmortemvisualization of the peritoneum shows nodular tumor masses (arrows; farright). FIG. 22C depicts Kaplan-Meier tumor-related survival curve oftumor-bearing NSG mice treated with either MOv19-BBζ or anti-CD19-BBζ Tcells via i.v. or i.p. injection. FIG. 22D depicts Kaplan-Meier overallsurvival of tumor-bearing NSG mice.

FIG. 23A-23B are a set of images and a plot showing that adoptivetransfer of FRα-specific T cells induces regression of ovarian cancerlung metastasis. NSG mice with 3 day established SKOV3 fLuc⁺ tumor inthe lungs received tail-vein injections of 6×10⁶ T cells expressingeither MOv19-BBζ or anti-CD19-BBζ on day 3 and day 8. FIG. 23A depictstumors, as monitored by BLI. FIG. 23B depicts quantified mean±SDbioluminescence signal photon emission from fLuc⁺ tumor cells.

FIG. 24 is a series of graphs showing that primary human T cellstransduced with MOv19-BBζ or MOv19-ζ preferentially produce Th1cytokines after stimulation with FRα+ cancer cell lines. Transduced Tcells (1×10⁵ CAR+ T cells) were cultured alone (none) or stimulatedovernight with an equal number of human FRα+ SKOV3 or antigen negativePEO-1 ovarian cancer cells. Cell free supernatant from three independentcultures was harvested and pooled after ˜20 hours of incubation, and theindicated human Th1/Th2 cytokines were quantified using cytometric beadarray technology. Values represent IFN-γ concentration (pg/ml) for theindicated cytokine.

FIGS. 25A-25C are a series of plots and images showing that primaryhuman T cells engineered to express FRα-specific CAR lyse FRα+ celllines in vitro. FIG. 25A depicts that the native mouse malignantmesothelioma cell line AE17 which does not express human FRα wastransduced to express high surface levels of human FRα (AE17.FRα) asshown by flow cytometry. Primary human T cells transduced to expresseither MOv19-ζ, MOv19-BBζ, MOv19-Δζ, or anti-CD19-BBζ CARs, or greenfluorescent protein (gfp) were co-cultured with Cr51-labeled native AE17or AE17.FRα cell lines for 4 hrs at the indicated effector to targetratio. FIG. 25B depicts the percent specific target cell lysis,calculated as (experimental−spontaneous release)±(maximal−spontaneousrelease) times 100. Results are expressed as mean of triplicate wellswith error bars indicating standard deviation. Human T cells transducedto express MOv19-ζ, MOv19-BBζ, MOv19-Δζ, or anti-CD19-BBζ CAR wereco-cultured at various effector to target ratios for 24 hrs with gfpexpressing AE17 or AE17.FRα cells. FIG. 25C depicts transduced cellsphotographed under fluorescent microscopy. Target cell lysis isindicated by imaging reduction in gfp-labeled adherent tumor cells.

FIGS. 26A-26D are a series of plots showing that tumor regression isassociated with the stable persistence of engineered human T cells invivo and dependent upon provision of CD137 costimulatory signaling. FIG.26A depicts tumor burden, as measured by averaged bioluminescent signal,per treatment group 4 weeks following T cell infusion. FIG. 26B depictsthe persistence of T lymphocytes in vivo assessed 4 weeks after transferof T cells expressing MOv9-BBζ delivered via i.v., i.t., or i.p. routesof administration or i.t. administration of T cells expressing MOv19-ζ,or control vectors (MOv19-Δζ or gfp; controls) by Trucount method. FIG.26C shows that four weeks after T cell therapy, the stable persistenceof engineered human T cells (x-axis) is negative correlated with thebioluminescent signal (y-axis; r=−0.78). Bcl-XL expression byFR-specific CAR CD8 T cells was examined after 3 days of culture inmedia alone (not shown) or with SKOV3. Bcl-XL expression waspreferentially increased in MOv19-BBζ CAR T cells populations (15.4%),compared with MOv19-ζ CAR+ T cells (6.7%) after stimulation with FRα+tumor cells. Culture in media alone did not induce Bcl-XL expression inCAR T cells. FIG. 26D depicts representative FACS analysis for one ofthree independent co-cultures.

FIG. 27 is a series of images depicting macroscopic evaluation ofresected tumor specimens following T cell therapy. Tumors were harvestedfrom NSG mice injected intratumorally (i.t.) with saline or T cellsbearing gfp, MOv19-Δζ, MOv19-ζ, MOv19-BBζ CARs; or injectedintravenously (i.v.) or intraperitoneally (i.p.) with MOv19-BBζ T cells.“No tumor” represents mice in which tumors were not detected. Tumorswere harvested from mice at the time of euthanasia, nearly 40 days afterfirst T cell injection.

FIG. 28 is an image depicting the study protocol schema for the clinicaltrial detailed elsewhere herein.

FIGS. 29A-29B are a set of plots showing that primary human T cellsengineered to express a fully-human anti-FR CAR containing the humanizedC4 scFv recognize and respond to FR expressing cancer cell lines invitro. scFv was efficiently expressed on the surface of T cellstransduced to express a first (−z) or second (−28z) CAR (FIG. 29A; usinga bicistronic vector for gfp co-expression). CAR transduced, but notuntransduced (UNT) T cells secreted IFN-g when co-cultured over nightwith ovarian or breast cancer cells expressing FR. Cell lines expressinglittle to no FR (A2780 and C30) were not recognized (FIG. 29B).

FIG. 30 is a table summarizing the identity of the SEQ ID NOs.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compositions and methods for treating cancerincluding but not limited to epithelial cancers. The present inventionrelates to a strategy of adoptive cell transfer of T cells transduced toexpress a chimeric antigen receptor (CAR). CARs are molecules thatcombine antibody-based specificity for a desired antigen (e.g., tumorantigen) with a T cell receptor-activating intracellular domain togenerate a chimeric protein that exhibits a specific anti-tumor cellularimmune activity.

The present invention relates generally to the use of T cellsgenetically modified to stably express a desired CAR. T cells expressinga CAR are referred to herein as CAR T cells or CAR modified T cells.Preferably, the cell can be genetically modified to stably express anantibody binding domain on its surface, conferring novel antigenspecificity that is MHC independent. In some instances, the T cell isgenetically modified to stably express a CAR that combines an antigenrecognition domain of a specific antibody with an intracellular domainof the CD3-zeta chain or FcγRI protein into a single chimeric protein.

In one embodiment, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain, a transmembrane domain, anda cytoplasmic domain. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Inanother embodiment, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.Preferably, the transmembrane domain is the CD8α hinge domain.

With respect to the cytoplasmic domain, the CAR of the invention can bedesigned to comprise the CD28 and/or 4-1BB (CD137) signaling domain byitself or be combined with any other desired cytoplasmic domain(s)useful in the context of the CAR of the invention. In one embodiment,the cytoplasmic domain of the CAR can be designed to further comprisethe signaling domain of CD3-zeta. For example, the cytoplasmic domain ofthe CAR can include but is not limited to CD3-zeta, 4-1BB and CD28signaling modules and combinations thereof. Accordingly, the inventionprovides CAR T cells and methods of their use for adoptive therapy.

In one embodiment, the CAR T cells of the invention can be generated byintroducing a lentiviral vector comprising a desired CAR targeting theα-folate receptor (αFR or FRα) into the cells. For example, thelentiviral vector comprises a CAR comprising anti-FRα, CD8a hinge andtransmembrane domain, and human 4-1BB and CD3zeta signaling domains,into the cells. The anti-FRα domain of the CAR of the invention can beany domain that binds to FRα including but not limited to monoclonalantibodies, polyclonal antibodies, antibody fragments, and humanizedantibodies. Therefore, as used herein, anti-FRα (or anti-αFR) refers toany composition targeted to FRα. The CAR T cells of the invention areable to replicate in vivo resulting in long-term persistence that canlead to sustained tumor control.

In one embodiment the invention relates to administering a geneticallymodified T cell expressing a CAR for the treatment of a patient havingcancer or at risk of having cancer using lymphocyte infusion.Preferably, autologous lymphocyte infusion is used in the treatment.Autologous PBMCs are collected from a patient in need of treatment and Tcells are activated and expanded using the methods described herein andknown in the art and then infused back into the patient.

The invention includes using T cells expressing an anti-FRα CARincluding both CD3-zeta and the 4-1BB costimulatory domain (alsoreferred to as FRα-specific CAR T cells). The FRα-specific CAR T cellsof the invention can undergo robust in vivo T cell expansion and canestablish FRα-specific memory cells that persist at high levels for anextended amount of time in blood and bone marrow. In some instances, theFRα-specific CAR T cells of the invention infused into a patient caneliminate cancerous cells in vivo in patients with epithelial ovariancancer. However, the invention is not limited to FRα-specific CAR Tcells. Rather, the invention includes any antigen binding moiety fusedwith one or more intracellular domains selected from the group of aCD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3zetasignal domain, and any combination thereof.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of 20% or ±10%, more preferably +5%, even more preferably+1%, and still more preferably +0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, the terms “FRα binding domain” may refer to any FRαspecific binding domain, known to one of skilled in the art. In oneexample, FRα binding domain comprises a single-chain variable fragment(scFv) comprising the variable regions of the heavy (V_(H)) and lightchains (V_(L)) of an antibody binding specifically to FRα. Anti-FRαantibodies, antibody fragments, and their variants are well known in theart and fully described in U.S. Patent Publications U.S 20100055034;U.S. 20090324594; U.S. 20090274697; U.S. 20080260812; U.S. 20060239910;U.S. 20050232919; U.S. 20040235108, all of which are incorporated byreference herein in their entirety. In one embodiment, the FRα bindingdomain is a homologue, a variant, an isomer, or a functional fragment ofan anti-FRα antibody. Each possibility represents a separate embodimentof the present invention.

As used herein, the terms “4-1BB (CD137) costimulatory domain” may referto any sequence of 4-1BB including, for example, a stimulatory signalingdomain of 4-1BB. Stimulatory signaling domains of 4-1BB and theirvariants are well known in the art and fully described in U.S. PatentPublication 20050113564, which is incorporated by reference herein inits entirety. Nucleic acid and amino acid sequences of 4-1BB and theirvariants are well known in the art and fully described in U.S. PatentPublications U.S. 20060063923; U.S. 20060029595; U.S. 20030082157; U.S.20020168719; U.S. 20040091476; U.S. 20050113564; and U.S. 20060002904,all of which are incorporated by reference herein in their entirety. Inone embodiment, the 4-1BB (CD137) costimulatory domain is a homologue, avariant, an isomer, or a functional fragment of 4-1BB (CD137). Eachpossibility represents a separate embodiment of the present invention.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. K and k light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologicall—competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell.

A “transfected” or “transformed” or “transduced” cell is one which hasbeen transfected, transformed or transduced with exogenous nucleic acid.The cell includes the primary subject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention provides compositions and methods for treatingcancer among other diseases. The cancer may be a hematologicalmalignancy, a solid tumor, a primary or a metastasizing tumor.Preferably, the cancer is an epithelial cancer, or in other words, acarcinoma. More preferably, the cancer is epithelial ovarian cancer.Other diseases treatable using the compositions and methods of theinvention include viral, bacterial and parasitic infections as well asautoimmune diseases.

In one embodiment, the invention provides a cell (e.g., T cell)engineered to express a CAR wherein the CAR T cell exhibits an antitumorproperty. The CAR of the invention can be engineered to comprise anextracellular domain having an antigen binding domain fused to anintracellular signaling domain of the T cell antigen receptor complexzeta chain (e.g., CD3 zeta). The CAR of the invention when expressed ina T cell is able to redirect antigen recognition based on the antigenbinding specificity. An exemplary antigen is FRα because this antigen isexpressed on malignant epithelial cells. However, the invention is notlimited to targeting FRα. Rather, the invention includes any antigenbinding moiety that when bound to its cognate antigen, affects a tumorcell so that the tumor cell fails to grow, is prompted to die, orotherwise is affected so that the tumor burden in a patient isdiminished or eliminated. The antigen binding moiety is preferably fusedwith an intracellular domain from one or more of a costimulatorymolecule and a zeta chain. Preferably, the antigen binding moiety isfused with one or more intracellular domains selected from the group ofa CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3zetasignal domain, and any combination thereof.

In one embodiment, the CAR of the invention comprises a CD137 (4-1BB)signaling domain. This is because the present invention is partly basedon the discovery that CAR-mediated T-cell responses can be furtherenhanced with the addition of costimulatory domains. For example,inclusion of the CD137 (4-1BB) signaling domain significantly increasedanti-tumor activity and in vivo persistence of CAR T cells compared toan otherwise identical CAR T cell not engineered to express CD137(4-1BB).

Composition

The present invention provides chimeric antigen receptor (CAR)comprising an extracellular and intracellular domain. The extracellulardomain comprises a target-specific binding element otherwise referred toas an antigen binding moiety. The intracellular domain or otherwise thecytoplasmic domain comprises, a costimulatory signaling region and azeta chain portion. The costimulatory signaling region refers to aportion of the CAR comprising the intracellular domain of acostimulatory molecule. Costimulatory molecules are cell surfacemolecules other than antigens receptors or their ligands that arerequired for an efficient response of lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

Antigen Binding Moiety

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding moiety. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenmoiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one embodiment, the CAR of the invention can be engineered to targeta tumor antigen of interest by way of engineering a desired antigenbinding moiety that specifically binds to an antigen on a tumor cell. Inthe context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder,” refers to antigens that are common tospecific hyperproliferative disorders such as cancer. The antigensdiscussed herein are merely included by way of example. The list is notintended to be exclusive and further examples will be readily apparentto those of skill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune responses. Theselection of the antigen binding moiety of the invention will depend onthe particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), j-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogeneHER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetalantigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD19, CD20 and CD37 areother candidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The type of tumor antigen referred to in the invention may also be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\yclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

In a preferred embodiment, the antigen binding moiety portion of the CARtargets an antigen that includes but is not limited to FRα, CD24, CD44,CD133, CD166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesteronereceptor, HER-2/neu, uPA, PAI-1, and the like.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen bindmoiety that is specific to the desired antigen target. For example, ifFRα, is the desired antigen that is to be targeted, an antibody for FRα,can be used as the antigen bind moiety for incorporation into the CAR ofthe invention.

In one embodiment, the antigen binding moiety portion of the CAR of theinvention targets FRα. Preferably, the antigen binding moiety portion inthe CAR of the invention is anti-FRα scFV, wherein the nucleic acidsequence of the anti-FRα scFV comprises the sequence set forth in SEQID: 3. In one embodiment, the anti-FRα scFV comprise the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 15. Inanother embodiment, the anti-FRα scFV portion of the CAR of theinvention comprises the amino acid sequence set forth in SEQ ID NO: 15.

In one embodiment, the antigen binding moiety portion in the CAR of theinvention is a humanized anti-FRα scFV, wherein the nucleic acidsequence of the humanized anti-FRα scFV comprises the sequence set forthin SEQ ID: 21. In one embodiment, the humanized anti-FRα scFV comprisethe nucleic acid sequence that encodes the amino acid sequence of SEQ IDNO: 23. In another embodiment, the humanized anti-FRα, scFV portion ofthe CAR of the invention comprises the amino acid sequence set forth inSEQ ID NO: 23.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

Preferably, the transmembrane domain in the CAR of the invention is theCD8 transmembrane domain. In one embodiment, the CD8 transmembranedomain comprises the nucleic acid sequence of SEQ ID NO: 5. In oneembodiment, the CD8 transmembrane domain comprises the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 17. Inanother embodiment, the CD8 transmembrane domain comprises the aminoacid sequence of SEQ ID NO: 17.

In some instances, the transmembrane domain of the CAR of the inventioncomprises the CD8 hinge domain. In one embodiment, the CD8 hinge domaincomprises the nucleic acid sequence of SEQ ID NO: 4. In one embodiment,the CD8 hinge domain comprises the nucleic acid sequence that encodesthe amino acid sequence of SEQ ID NO: 16. In another embodiment, the CD8hinge domain comprises the amino acid sequence of SEQ ID NO: 16.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention is responsible for activation of at leastone of the normal effector functions of the immune cell in which the CARhas been placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can bemediated by two distinct classes of cytoplasmic signaling sequence:those that initiate antigen-dependent primary activation through the TCR(primary cytoplasmic signaling sequences) and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the invention comprises a cytoplasmicsignaling sequence derived from CD3 zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR of the invention. For example, the cytoplasmic domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention in exemplified primarily with 4-1BB as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. Inanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of 4-1BB. In yetanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

In one embodiment, the cytoplasmic domain in the CAR of the invention isdesigned to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises thenucleic acid sequence set forth in SEQ ID NO: 6 and the signaling domainof CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO:7.

In one embodiment, the cytoplasmic domain in the CAR of the invention isdesigned to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises thenucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:18 and the signaling domain of CD3-zeta comprises the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 19.

In one embodiment, the cytoplasmic domain in the CAR of the invention isdesigned to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises theamino acid sequence set forth in SEQ ID NO: 18 and the signaling domainof CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:19.

Vectors

The present invention encompasses a DNA construct comprising sequencesof a CAR, wherein the sequence comprises the nucleic acid sequence of anantigen binding moiety operably linked to the nucleic acid sequence ofan intracellular domain. An exemplary intracellular domain that can beused in the CAR of the invention includes but is not limited to theintracellular domain of CD3-zeta, CD28, 4-1BB, and the like. In someinstances, the CAR can comprise any combination of CD3-zeta, CD28,4-1BB, and the like.

In one embodiment, the CAR of the invention comprises anti-FRα scFv,human CD8 hinge and transmembrane domain, and human 4-1BB and CD3zetasignaling domains. In one embodiment, the CAR of the invention comprisesthe nucleic acid sequence set forth in SEQ ID NO: 1. In anotherembodiment, the CAR of the invention comprises the nucleic acid sequencethat encodes the amino acid sequence of SEQ ID NO: 13. In anotherembodiment, the CAR of the invention comprises the amino acid sequenceset forth in SEQ ID NO: 13.

In one embodiment, the CAR of the invention comprises humanized anti-FRαscFv, human CD8 hinge and transmembrane domain, and human 4-1BB andCD3zeta signaling domains. In one embodiment, the CAR of the inventioncomprises the nucleic acid sequence set forth in SEQ ID NO: 20. Inanother embodiment, the CAR of the invention comprises the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 22. Inanother embodiment, the CAR of the invention comprises the amino acidsequence set forth in SEQ ID NO: 22.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

Activation and Expansion of T Cells Whether prior to or after geneticmodification of the T cells to express a desirable CAR, the T cells canbe activated and expanded generally using methods as described, forexample, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869;7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; andU.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besangon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof, and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (Tc, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of Tc cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of Tc cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) transduced witha lentiviral vector (LV). For example, the LV encodes a CAR thatcombines an antigen recognition domain of a specific antibody with anintracellular domain of CD3-zeta, CD28, 4-1BB, or any combinationsthereof. Therefore, in some instances, the transduced T cell can elicita CAR-mediated T-cell response.

The invention provides the use of a CAR to redirect the specificity of aprimary T cell to a tumor antigen. Thus, the present invention alsoprovides a method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal comprising the step ofadministering to the mammal a T cell that expresses a CAR, wherein theCAR comprises a binding moiety that specifically interacts with apredetermined target, a zeta chain portion comprising for example theintracellular domain of human CD3zeta, and a costimulatory signalingregion.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellis able to kill tumor cells in the recipient. Unlike antibody therapies,CAR T cells are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In one embodiment, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. For example, it was unexpectedthat the FRα-specific CAR T cells of the invention can undergo robust invivo T cell expansion and persist at high levels for an extended amountof time in blood and bone marrow and form specific memory T cells.Without wishing to be bound by any particular theory, CAR T cells maydifferentiate in vivo into a central memory-like state upon encounterand subsequent elimination of target cells expressing the surrogateantigen.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified T cells may be an activeor a passive immune response. In addition, the CAR mediated immuneresponse may be part of an adoptive immunotherapy approach in whichCAR-modified T cells induce an immune response specific to the antigenbinding moiety in the CAR. For example, FRα-specific CAR T cells elicitan immune response specific against cells expressing FRα.

While the data disclosed herein specifically disclose lentiviral vectorcomprising anti-FRα scFv, human CD8a hinge and transmembrane domain, andhuman 4-1BB and CD3zeta signaling domains, the invention should beconstrued to include any number of variations for each of the componentsof the construct as described elsewhere herein. That is, the inventionincludes the use of any antigen binding moiety in the CAR to generate aCAR-mediated T-cell response specific to the antigen binding moiety. Forexample, the antigen binding moiety in the CAR of the invention cantarget a tumor antigen for the purposes of treat cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

In one embodiment, the antigen bind moiety portion of the CAR of theinvention is designed to treat a particular cancer. FRα is aglycosylphosphatidylinositol-anchored protein that is overexpressed onthe surface of cancer cells in a spectrum of epithelial malignancies,but is limited in normal tissue. As such, CARs designed to target FRαcan be used to treat any disease or disorders, including but not limitedto epithelial cancers, characterized by cells and/or tissues displayingan overexpression of FRα. For example, the CAR designed to target FRαcan be used to treat cancers and disorders including but are not limitedto ovarian cancer, lung cancer, breast cancer, renal cancer, colorectalcancer, other solid cancers and the like.

However, the invention should not be construed to be limited to solelyto the antigen targets and diseases disclosed herein. Rather, theinvention should be construed to include any antigenic target that isassociated with a disease where a CAR can be used to treat the disease.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of ovarian cancer. Incertain embodiments, the cells of the invention are used in thetreatment of patients at risk for developing ovarian cancer. Thus, thepresent invention provides methods for the treatment or prevention ofovarian cancer comprising administering to a subject in need thereof, atherapeutically effective amount of the CAR-modified T cells of theinvention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

The materials and methods employed in these experiments are nowdescribed.

Generation of Anti-α Folate Receptor (αFR) T-Body Molecules

The anti-αFR scFv (MOv19) was used as a template for PCR amplificationof an 780-bp MOv19 fragment using the following primers:

(SEQ ID NO: 24) 5′GCGGGATCCTCTAGAGCGGCCCAGCCGGCCATGGCCCAGGTG-3′(BamHI is underlined) and (SEQ ID NO: 25)5′GCGGCTAGCGGCCGCCCGTTTTATTTCCAACTTTGTCCCCCC-3′ (NheI is underlined).

The resulting PCR product contained a BamHI site on the 5a end and aNheI site on the 3α end. The CD8a hinge, transmembrane, and cytosolicregions were amplified by PCR using previously constructed templates andthe following primers:

(SEQ ID NO: 26) 5′GCTGGGACAAAGTTGGAAATCAAAGCTAGCACCACGACGCCAGCGCCGCGACC-3′ (NheI is underlined) and (SEQ ID NO: 27)5′TCGACAGTCGACTTAGCGAGGGGGCAGGGCCT-3′(for the functional TCRζ containing molecules, SalI is underlined).

The chimeric immunoreceptor constructs were generated through genesplicing by overlap extension. Equimolar amounts of the MOv19 PCRproduct and CD8 hinge, transmembrane, and cytosolic PCR products werecombined with

(SEQ ID NO: 28) 5′ATAGCATCTAGAATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTC-3′ (XbaI is underlined) and (SEQ ID NO: 29)5′TCGACAGTCGACTTAGCGAGGGGGCAGGGCCT-3′(for the functional TCRζ containing molecules, SalI is underlined).

The final PCR products were then digested with XbaI and SalI and ligatedinto pELNS, a third generation self-inactivating lentiviral expressionvectors based on pRRL-SIN-CMV-eGFP-WPRE in which transgene expression isdriven by the EF-1α promoter, which replaced the CMV promoter.High-titer research grade replication-defective lentiviral vectors wereproduced and concentrated.

The investigational agent in this protocol is autologous T cellstransduced with α-FR-CAR (FIG. 6). Autologous T cells are transducedwith a lentiviral vector expressing the α-FR CAR. This redirectsspecificity of the transduced T cells for tumor that expresses αFR,which is expressed at high levels in 90% of epithelial ovarian carcinoma(EOC) but is largely absent from normal. The α-FR CAR, is linked to anintracellular signaling molecule comprised of the TCRζ, 4-1BB. The scFvMOv19 is derived from a mouse monoclonal antibody, and thus containsmouse sequences that are immunogenic. If early clinical feasibility andefficacy is established, this scFv is humanized for later stage clinicaldevelopment. The cytoplasmic signaling domains of the transgene areentirely of the native human sequences. These receptors are “universal”in that they bind antigen in an MHC-independent fashion, thus, onereceptor construct can be used to treat a population of patients withalpha folate receptor antigen-positive tumors. The final transgeneconstruct was cloned into the pELNS lentiviral vector (FIGS. 6 and 7).

The plasmids used for alpha folate receptor (αFR) chimeric immunereceptor genes delivery are schematically depicted in FIG. 8A. Thetransfer vector is an HIV derived self inactivating (SIN) vector thatcomprises a 5′ LTR and a 3′ U3 deleted LTR. Transgene transcription isdriven off the mammalian ef-1α promoter. The transgene is composed ofextracellular domain MOv19 (αFR scFv) and signaling domain 4-1BB andCD3zeta chain. The vector also contains the central polypurine tract andcentral termination sequence (cppt/CTS) for improved transductionefficiency, the rev response element (RRE) for RNA transport, the WPREelement for improved RNA translation and, the packaging sequence.

Novel CARs were constructed that contain a FRα-specific scFv (MOv19)coupled to either an inactive form of the CD3-ζ intracellular domain(MOv19-Δζ), CD3 ζ chain signaling module (MOv19-ζ) or in combinationwith the CD137 (4-1BB) costimulatory motifs (MOv19-BB-ζ) (FIG. 6). HumanT cells were transduced with the CAR using lentivirial vectors. Inco-culture assays, CAR transduced T cells were measured for reactivityagainst ovarian cancer cells expressing FRα via IFN-g ELISA and cytokinebead assay. Cytotoxicity was measured using a bioluminescence system invitro. The potential antitumor efficacy of αFR CAR in Winn assay andxenograft model in NOG mice was explored.

Preparation, Structure, and Composition of the Materials that can beGiven to the Patients or Used to Treat the Patient's Cells.

The CAR constructs were developed at the University of Pennsylvania, andthe clinical grade vectors were cloned and are manufactured at The Cityof Hope under the direction of Dr. Larry Couture. The clinical gradeengineered T cells are manufactured in the Clinical Cell and VaccineProduction Facility at the University of Pennsylvania. At the end ofcell cultures, the cells are cryopreserved in infusible cryomedia inbags. Each bag contains an aliquot (volume dependent upon dose) ofcryomedia containing infusible-grade reagents. Subjects in Stratum 1 canreceive a single dose of α-FR CAR transduced T cells by directintratumoral injection, either ultrasonically guided or intraoperativelyusing a dose escalation approach. Subjects in Stratum 2 receive splitdose intravenous infusions (10%, 30%, and 60% on days 0, 1, and 2,respectively) of the transduced T cells using a dose escalationapproach.

Measuring DNA Purity

The DNA used to manufacture the vector is isolated from E. coli cellsgrown in LB medium under ampicillin selection. The DNA undergoes qualitycontrol (QC) release testing to ensure its identity and purity. DNA istested for appearance (clear and odorless), 260/280 ratio (1.7-2.0),agarose gel electrophoresis (≥90% supercoiled), residual RNA (nondetected/λg), linear plasmid DNA or chromosomal DNA (none detected/μg),restriction enzyme mapping, endotoxin (<30 EU/mg), and sterility.

Viral Production

Lentiviral vector is produced by City of Hope. City of Hope has built aPhaseI/II cGMP Manufacturing facility consisting of two (2),independent, Class 10,000 manufacturing Suites (A&B) inside aunidirectional GMP support area. Each Suite consists of: anEntry/Gowning Airlock, a Cell Expansion Lab, Production Lab,Purification/Buffer Prep Lab, and Exit/Degowning Airlock. Each Suite hasan independent air handler with HEPA supply and controlled exhaust.Pressure differentials between suites and within suites is controlled aswell as temperature and humidity. Environmental conditions, and GMPequipment, is monitored, recorded and alarmed through a centralizedBuilding Monitor System. All transfers and any open vessel operationstake place in the suites in unidirectional flow, Class 100, hoods.

Supporting GMP areas include: GMP Controlled Storage, −80° C. ControlledStorage, +4° C. Controlled Storage, USP Water System, USP GasDistribution Systems, Materials Airlock, separate locker room, GowningAirlock, Entry Hallway, Autoclave/Wash Room, and Exit Hallway. AllAirlocks, hallways, and direct access support areas are supplied bymultiple GMP air handlers with HEPA filtered supply air to supportspaces with an overall Classification of 100,000.

Viral Production in 293T Cells

Viral vector is produced by transient transfection using a three plasmidproduction system that provides gag/pol, tat, rev, and VSV-G in a systemsimilar to (Zufferey et al, 1997). The only regions of overlap are inthe packaging and gag and cppt/CTS sequences. RCL testing is performedin accordance with FDA guidelines as described later.

The process begins with a single vial of frozen, 293T cells from a GMP,validated, Master Cell Bank. Cells are thawed and expanded through anumber of passages to increase cell count and volume of cells in anumber of sizes of culture flasks. The City of Hope has both adherentand suspension cell lines. This is a general description of the adherentcell line production process:

The thawed cells are centrifuged to a pellet, resuspended in cellculture medium and a cell count is performed. Multiple flasks containingmedium are seeded with cells to a specific cell density. The flasks areincubated at 37° C. and 5% CO₂ concentration in an incubator for ˜24hours. After 24 hours the medium is removed from the flasks containingcells, and Growth Media is added to the flasks and cells. Flasks areincubated again at same temperature and CO₂ for an additional 24 hours.Flasks are removed from the incubator, media removed, cells are loosenedfrom the surface with Trypsin solution, cells counted, and expanded to 5times more flasks to a specified seeding density with Growth Media inflasks. Flasks are incubated at 37° C. and 5% CO₂ for 48 hours. At theend of that time, media is removed from the flasks, cells aretrypsinized, cells counted, and up to 10 Cell factories (4 layer) areseeded to a specified cell density and 800 mL of Growth Media per cellfactory. Cell factories are incubated at 37° C. and 5% CO₂ forapproximately 72 hours. Media is then removed from the Cell factories,cells are trypsinized, counted, and used to seed ten (10) Cell factories(10 layer each) with approximately 1.5 L of Growth Media. These ten (10)cell factories are incubated at 37° C. and 5% CO₂ for ˜ 24 hours. Themedia is removed from the cell factories and the cells are transfectedwith Transfection reagent containing media, transfection reagent andthree GMP plasmids.

The Transfection reagent consists of three GMP produced plasmids; theDNA plasmid containing effective gene, and two DNA helper plasmids, p93and pVSV-G, in a calcium phosphate solution. The GMP plasmids areprepared by fully sequencing the DNA plasmid, transfecting an E. colistrain with the DNA, and growing the E. coli to produce a Master CellBank. A vial from the Master Cell Bank is used to express the DNAplasmid which is purified, aliquoted, and tested for sequence, purity,and sterility.

After the cell factories are transfected with Transfection reagent theyare incubated for approximately 20 hours. The transfection reagent ispoured off and Growth media is added to the cell factories. They arethen incubated for an additional 24 hours before harvest.

Vector Purification and Release Testing

Viral vector is purified in compliance with good manufacturing practices(GMP). The fluids containing vector are poured from the cell factories,pooled, and then filter clarified through a 0.8/0.45 micronclarification filter. The clarified harvest is loaded on to an anionexchange resin in a chromatography column. The ion exchange column iswashed with a low salt buffer and then the semi-purified vector iseluted with a 0.7 M NaCl buffer.

The elution buffer is then concentrated using a 500 kD tangential flowfilter (TFF) membrane and diafiltered with a low salt buffer to aconcentration of 10×. A benzonase endonuclease is added to the solutionto remove residual DNA and incubated for approximately 1 hour. Thetreated solution is further concentrated with the 500 kD TFF membranefor an additional 10×, and then diafiltered with the formulation bufferwith approximately ten (10) volumes. Following the diafiltration, thepurified vector solution is further concentrated from 10× to 20× asrequired. The final concentrate is filled into sterile plasma bags withthe desired volume, sampled, and then frozen to −80° C.

Samples are tested, and all documentation is reviewed, and approvedbefore the material is released. Vector release testing is performed asfollows: purity is tested by visual inspection (clear and colorless), pH(7.0-7.4), conductivity (4-7 mS/cm), fill volume (≥40 ml), total protein≤0.70 mg/ml and benzonase (≤100 ng/ml or 0.1 ppm); identity is tested bysilver stained SDS-page and by RT-PCR specific for theconstruct/transgene; potency is tested by titer on 293 cells (≥2.5×10⁷TU/ml); for safety, the vector is tested for gag DNA by qPCR(undetectable), and for RCL by VSV-G DNA and a biological RCL test(described below); sterility testing includes endotoxin (<100 EU/ml),sterility (no growth), adventitious virus (undetectable) and mycoplasma(undetectable); vector is also tested by p24 ELISA (0.1-g/ml). Aseparate C of A can be provided for each vector.

RCL Assay

Testing for recombination or replication competent lentivirus (RCL) isperformed in accordance with FDA guidelines for the testing for RCR.C8166 cells are exposed to vector supernatant and passaged for 3 weeks.Culture supernatants are monitored for p24 production by ELISA, and forpersisting or increasing numbers of packaging DNA measured by PCR. Thetest article for these assays, in accordance with the guidelines, is 5%or 300 ml of culture supernatant, whichever is less and 10'10⁸ end ofproduction (EOP) cells. Testing is performed at the National Gene VectorLaboratories at Indiana University.

Packaging, Shipment and Storage of the Vector

The first lots of vector is shipped to UPENN in accordance with City ofHope shipping SOPs as described in the Lentigen DMF. Shipment of futurevector lots may occur from Omnia Biologics, Indiana University, or fromLentigen Corporation, depending on where the GMP lot is manufactured.

Vector Stability Monitoring Plan

The potency of the vector is determined in each cell product bytransduction efficiency as measured by copy number and/or transgeneexpression levels. A stability monitoring plan is not be put in placefor the vector for this study at this stage because each vector lot isindividual and small in size. In addition, a stability testing planrequires that the CVPF ship vector back to the manufacturing facilityfor titration using their standard assay. The introduction of a shippingstep to the stability testing introduces a variable.

Intended Target Cells of Recombinant DNA, Cells that are to be TreatedEx Vivo and Returned to the Patient, Characterized of Cells Before andAfter Treatment, and Target Cells Incorporation of the DNA

The target cell product is autologous CD3+ autologous T lymphocytes. Tlymphocytes are enriched from a leukapheresis product by depletion ofmonocytes via counterflow centrifugal elutriation on the Gambro Elutra,which employs a single use closed system disposable set. On day 0, theαFR T-body manufacturing process is initiated with activation of Tlymphocytes with anti-CD3/CD28 mAb coated magnetic beads. αFR vector isadded in split doses with 50% on day 0 and 50% on day 1. Vectortransduction occurs between days 0 and 3. On day 3, the cells are washedand media is replaced. Cultures are typically expanded from 9-12 days.At culture harvest, cells are depleted of magnetic beads, washed,concentrated, and cryopreserved.

At the start of culture, the enriched CD3+ T-cell culture generallycontains some amount of residual αFR CAR+ cells (B cells ˜5-10%), CD16+(NK cells ˜5-10%) and CD14+ cells (macrophages at ˜<5%). Therefore,these cells are exposed to the vector during transduction, andincorporate the recombinant transcript. After expansion in vitro, thefinal cellular product is typically >90% CD3+ lymphocytes. Cultureconditions don't support growth of macrophages or B cells, and by theend of the culture period B cells comprise ˜<2%, and macrophages ˜<1% ofthe total culture.

The method of ex-vivo transduction ensures that only peripheral whiteblood cells enriched for lymphocytes are exposed to the vector. Anyresidual non-integrated vector is washed away at day 3 during theexpansion and again during the harvest and concentration prior toformulation of the final cellular product. The vector cannot mobilize,even in the presence of HIV, accordingly, there are no concernsregarding vertical or horizontal transmission of the vector, ortransmission to cells not present in the starting culture. Below is adetailed description of the manufacturing process:

Cell Collection and Purification

On day 0 of the T-cell manufacturing process, non-mobilized peripheralblood leukocytes is obtained through a leukapheresis collection.Approximately 10 L is collected and processed on the Baxter Amicus CellSeparator or equivalent to obtain a population of approximately 5-15×10⁹white blood cells. The product is taken to the CVPF, where samples aretaken for bacterial and fungal cultures and phenotyping by flowcytometry. Cell number is determined on the Coulter Multisizer III andviability is tested by trypan blue exclusion assay. The apheresisproduct is then processed with the Gambro Elutra, which utilizescounter-flow centrifugal force to separate cell populations based onsize and density. The Elutra operates as a closed system and the use ofa disposable tubing set further minimizes the risk of contamination. Thelymphocyte fractions collected following the Elutra separation arecombined and washed using the Baxter CytoMate, Cell Saver 5 or the COBE2991 Cell Processor. Composition of the cell product (CD4+, CD8+, B-celland macrophages, etc) is assessed and tracked by flow cytometryperformed on the post-Elutra lymphocyte fraction, and the monocytefraction.

Following the cell enrichment process (day 0), the enriched lymphocytesare activated with anti-CD3/anti-CD28 mAb coated magnetic microbeads ata 3:1 bead to cell ratio. This optimal bead: cell ratio was previouslydetermined (IND #6675 and Levine et al., 1997). Enriched lymphocytes(via elutriation or positive selection) are stimulated with Dynabeadsconjugated with mouse anti-human CD3 and CD28 in static tissue cultureflasks. Transduction is performed on day 0 and day 1 of culture with apredetermined MOI (25 TU/cell of the FR vector for example) by additionof the αFR CAR lentiviral vector at 50% of the total transduction MOIper day. The vector is washed away by media replacement in a BaxterCytoMate Cell Processing System or Cell Saver 5 device on day 3 ofculture. After the vector wash off, the cell and bead mixture are seededback into gas permeable tissue culture flasks in fresh media and placedat a 37° C. incubator with 5% CO₂ and >90% of humidity for cultivationand further expansion. The cell culture is maintained in a closedsystem. Tubing leads on the tissue culture flasks are connected ordisconnected through a variety of sterile tubing connecting devices andheat sealers (e.g. spike connectors, tubing welds from the TerumoSterile Connecting Device, heat seal from the Sebra Heat Sealer) toprevent the risk of contamination. Cells are counted daily from day 3 today 5 of culture. After 5 days of cultivation, cells may be countedevery other day. Fresh media is added to the culture to maintain thecells at an appropriate density. During the log phase of cell growth, ifneeded, cultures are transferred to the WAVE Bioreactor, where cellconcentrations may reach 1×10⁷ cells/ml or higher. Optimized cellculture conditions in both the WAVE Bioreactor 2/10 and 20/50 has beenpreviously established (June IND 12799 and Hami et al., 2003, 2004). Theadvantage of the WAVE is that T cells are grown at higher densities,which saves labor on processing and during the cell harvest. For celldoses up to 1×10¹⁰ the WAVE Bioreactor is not needed.

Culture Harvest and Final Formulation, Cryopreservation

On the final day of the culture, cells are harvested and concentratedusing the Baxter Fenwal Harvester or an equivalent system. Before andafter processing cells through the Harvester, the cell product is placedon the Baxter MaxSep for removal of the anti-CD3/CD28 magneticmicrobeads. αFR CAR T cells are resuspended in cryopreservation media.Cells are frozen in bags using a controlled-rate freezer. CryopreservedαFR CAR T cell products are stored in a monitored freezer at ≤−130°C.Each infusion bag can contain ˜10-50 mL of cells depending upon thesize of the dose.

Cell Purity and Release Testing

Release testing for αFR-CAR T cells is similar to previous similar genetherapy cellular products manufactured for clinical trials at theClinical Cell and Vaccine Production Facility (ref June IND #6675, 8568,12799, 13911). The αFR-CAR T cells are tested and released based on cellviability (sentinel tube, ≥70%), % CD3+ cells (≥80%), residual beadcount (≤100 beads/3 million cells), endotoxin (<3.5 EU/ml; the limit of3.5 EU/ml was derived in the following manner: at a max volume of 100mls, cryopreserved cell concentration of 100×10⁶/ml equals a cell doseof 1×10¹⁰ cells. 3.5×100=350 EU, avg person=70 kg, so limit is 5 EU/kg),vector copy number (≥0.2 copies per cell), αFR CAR expression (≥20%expression by flow cytometry), VSV-G DNA (undetectable), RCL by HIVgagDNA and p24 protein (negative), mycoplasma (negative), andbacteria/fungal cultures (no growth).

Structure of the Added DNA Sequences Monitored and Sensitivity of theAnalysis.

Lentiviral vectors permanently modify the cell's DNA by integrating aDNA copy of their viral genome into cellular DNA. Thus, these sequencescan be monitored in vivo by PCR of DNA isolated from PBMCs. A PCR assayhas been developed that detects the junction region between the CARchimeric signaling domain, so that the genetic construct can bedistinguished from the endogenous signaling chains that exist in nature.This allows for monitoring of the persistence of the vector modifiedcells in each patient.

Stability of the Added DNA Both in Terms of its Continued Presence andits Structural Stability.

For safety purposes, the average culture copy number per cell is limitedto the range of 0.2-5. The copy number after expansion is stable sincethe vector is stably integrated into the cell's DNA. Detection of vectorcopies in vivo after dosing may increase or decrease over time, as aresult of the expansion, trafficking and persistence of the vectormodified cells.

The results of the experiments are now described.

Example 1: Eradication of Human Ovarian Cancer Following AdoptiveTransfer of Genetically Modified α-Folate Receptor-Specific T Cells

Human T cells expressed cell surface scFv Mov19 40˜50% after lentiviraltransduction (FIGS. 3A-3B). MOv19-ζ and BB-ζ transduced T cellsdemonstrated target-specific release of IFN-g, TNF-α and IL-2 cytokinesand cytotoxicity function when co-cultured with FRα+ tumor cells, whileT cells transduced with MOv19-Δζ or with GFP did not (FIGS. 4A-4C). Inan in vivo Winn assay, MOv19-ζ transduced T cells were able to inhibitthe out growth of FRα+ ovarian cancer (FIGS. 5A-5E and 6). In contrast,T cells transduced with the MOv19-Δζ or with GFP had no effect on tumorgrowth (FIGS. 5A-5E and 6). Notably, in vivo anti-tumor activity ofMOv19 CAR was improved through provision of 4-1BB (CD137) signaling(FIG. 6). Furthermore, it is demonstrated that incorporation of thecostimulatory domains enhanced the persistence of T cells and isassociated with improved anti-tumor efficacy in vivo (FIG. 7).

Example 2: Efficiency of DNA Delivery and Target Cell PercentageContaining DNA

The lentiviral vector delivery system described is highly efficient indelivery of genes to T lymphocytes. In preclinical experiments using theT-body constructs proposed for this trial, it was routinelydemonstrated >60% transduction efficiency for the α-FR-CAR protein inCD4+ T cells transduced to express the αFR CAR with CD3 zeta and 4-1BBsignaling domains (FIG. 8B). Lentiviral vectors are well known for theirsuperior gene transfer efficiency when compared to murine retroviralvector transduction efficiencies.

Example 3: In Vitro Assessment of Function

Several preclinical studies have been carried to demonstrate the invitro and in vivo efficacy of the gene transfer system and its payload(FIGS. 9A-9C). To investigate the antitumor potential of the transducedT cells, effector function was measured in standard chromium releaseassays using αFR-negative AE17 cells, AE17.FR (a derivative engineeredto express αFR), and established human ovarian cancer cell lines. Tcells transduced with αFR CAR efficiently lysed AE17.FR but did not killparental AE17 cells. Importantly, the αFR CAR-transduced T cells werealso highly cytotoxic for carcinoma cells that express αFR, killinghuman ovarian cancer SKOV3 cell lines. The inclusion of 4-1BB (CD137)costimulatory domains in tandem or in triplicate with TCR-ζ generallydid not increase in vitro cytotoxicity above that of T cells expressingαFR CAR TCR-ζ only. The killing was efficient, with plateau lysisoccurring at a 10:1 E:T ratio during a 20-h culture (FIG. 10) suggestingthat the redirected T cells were capable of serial killing. Moreover,the lysis was specific because T cells transduced with GFP or anirrelevant CD19-CAR showed no cytotoxic activity against the same targetcells, excluding alloreactivity or nonspecific lysis. Furthermore, Tcells expressing a truncated TCR-ζ intracellular domain (αFR-Δz) alsofailed to kill αFR-expressing targets, demonstrating the requirement foran intact TCR-ζ signaling domain. CAR+ T cells were co-incubated with apanel of tumor cell lines and the amount of secreted effector cytokineIFN-g was determined. CAR+ T cells recognized FRα+ tumor lines SKOV3 andA1847 and secreted IFN-g at very high levels. A moderate level of IFN-gwas observed in co-incubated with the OVCAR3 and A2780, which expressFRα at moderate level. A low level of IFN-g was observed in co-incubatedwith the C30 and PEO-1 cell line, which was negative for αFR expressionby FACS analysis (FIG. 9C).

Example 4: In Vivo Assessment of Function Winn Assay

As an initial test of in vivo antitumor activity of the αFR CARconstructs, Winn assay was performed by the s.c. injection of a αFR+human ovarian cancer cell line SKOV3 expressing Luciferase (1×10⁶cells/mouse) mixed with CAR expressing T cells (1×10⁶ cells/mouse). Theanimals were imaged after inoculation and every 10 days to evaluatetumor growth, and photon emission from luciferase-expressing cells wasquantified using the “Living Image” software (Xenogen). No effect wasobserved on the tumors growth in mice treated with either GFP or αFRCARdz transduced T cells (FIG. 11A). In these control groups, animalsstarted to develop a tumor at approximately day 30 after inoculationwith all animals developing tumor by day 40 (FIG. 11B). By contrast,mice injected with αFR CAR-z or BBz bearing T cells inhibited tumoroutgrowth equally until day 30 after inoculation demonstrating arequirement for an intact TCR CD3 zeta signaling domain in Winn assay.However, after another 10 days, all mice from αFR CAR CD3 zeta group haddetectable tumor (4/4), whereas only 3/5 mice from the BBz group hadtumors. These tumors are substantially smaller than the CD3zeta grouptumors. These data show that incorporation of 4-1BBζ CD137) into FRα CARcould enhance anti-tumor activity in vivo.

Example 5: Xenograft Model

To further explore the potential antitumor efficacy of the αFR-CARconstructs, a xenograft model using SKOV3 Luc tumor cells was developed.6 to 12-week-old female NOG mice were inoculated s.c. with 3×10⁶ SKOV3Luc cells on the flank on day 0. After tumors become palpable about 1month, human primary T cell (CD4+ and CD8+ T cells used were mixed at1:1 ratio) were activated, and transduced as described above. After 2weeks T ell expansion, when the tumor burden was 200-300 mm³, the micewere treated with T cells (˜40%-50% transgene-positive). The route,dose, and timing of T-cell injections is indicated in the individualfigure legends. Tumor dimensions were measured with calipers, and tumorvolumes calculated using the formula V=¹2 (length×width²), where lengthis greatest longitudinal diameter and width is greatest transversediameter. Tumor-bearing mice were treated with intratumoral injectionsof 20×10⁶ T cells (˜40%-50% transgene positive) on day 40 and 45 posttumor inoculation. Human donors were used to generate the transduced Tcells. All the mice in the saline group, which did not receive cellbased therapy, showed continued tumor growth. Similarly, the micereceiving αFR CAR dz with signaling deficiency or GFP transduced T cellsshowed continued tumor growth beyond the time of T cell transfer. Themice receiving αFR CAR-z T cells showed slowed tumor growth which was nosignificantly different when compared to all three control group (FIG.12 A, C). The mice receiving αFR CAR BBz T cells displayed rapid tumorregression compared to all other the groups, suggesting that 4-1BBsignaling mediate enhanced antitumor responses in vivo.

In order to evaluate the effect of different routes of administration,the tumor bearing mice were also treated using αFR CAR BBz transduced Tcells by intravenous (i.v.), intraperitoneal (i.p.) injection andintratumoral (i.t.) injection. Following i.v. and i.p. injections, apotent antitumor effect was again observed (FIG. 12 B,D), but showedabout 7 days delayed reduction in tumor mass compare to the intratumoralroute of administration (FIG. 12 B). The intratumoral injection appearsto be the superior route of administration, marginally faster than i.v.and i.p.

Example 6: Persistence of Human T Lymphocytes after Transfer

Next, the persistence of the engineered T cells in all mice wasdetermined. Peripheral blood was obtained on day 74, 4 weeks after thelast adoptive T cell transfer, and quantified for the presence of CD4and CD8 T cells. The CD4+ and CD8+ T cell counts were highest in miceafter injection with BBz CAR+ T cells by IT, IP and IV route compared togfp, αFR CAR dz and the CD3zeta group (FIG. 13). Notably, the counts ofCD4+ and CD8+ T cells in BBz group was significantly higher than z group(P<0.01), while the total T cell counts in the z group is similar withother control groups including saline group without T cells injection(p>0.05).

Example 7: Antigen Specific Model

Having shown that αFR CAR containing 4-1 BB mediate enhanced survive ofT cells and increased anti-tumor activity in vivo, it was next sought todetermine whether the αFR CAR anti-tumor activity is antigen specific. ACD19 specific CAR also containing 4-1BB singling domain was evaluated inthe xenograft model. On week 6 after establishing the tumor,tumor-bearing mice were treated with intratumoral injections of 20×10⁶ Tcells (˜40%-50% transgene positive) on day 40 and 45. Followingtreatment, a rapid reduction in tumor mass was observed in αFR CAR BBzgroup (FIG. 14A). In contrast, the tumor grew progressively in micetreated with T cells expressing GFP or CD19 CAR group. Thus, αFR CAR BBzeradication of SKOV3 tumor is antigen-specific because the CD19 CAR alsocontaining a 4-1BB co-stimulatory signaling domain displayed noantitumor activity (FIGS. 14A-14B). Mice treated with intratumoral αFRCAR BBz had significantly higher (P<0.05) T-cell counts than theintratumoral anti-CD19 group, suggesting that tumor antigen drives theexpansion of the adoptively transferred T cells in vivo (FIG. 14C).

Example 8: Intraperitoneal Model of Human Ovarian Cancer

In the previous experiment, it was demonstrated that local injection ofCAR T cells results in eradication of established tumor in vivo. It wasfurther determined the antitumor activity of αFR-specific T cells in anintraperitoneal model, because ovarian cancer is a disease usuallyconfined to the peritoneal cavity. After 30 days, IP inoculation 5×10⁶SKOV3Luc cells efficiently produced peritoneal carcinomatosis (FIG.15.C). A swollen abdomen, indicative of ascites formation and heavilyperitoneal carcinomatosis, was observed within 1 to 3 weeks after Tcells expressing CD19 CAR BBz transfer via IV or IP route (FIG. 15.A).These mice developed marked bloody ascites (5-8 ml) and multiple nodularperitoneal tumors and had to be euthanized within 1 to 3 weeks after Tcell injection due to abdominal distention. While all the mice treatedwith αFR CAR BBz did not form ascites and exhibited enhanced survival(FIG. 15B.). 60% (3/5) and 40% (2/5) mice bearing SKOV3 tumor remainalive by 10 weeks following αFR CAR BBz T cell transfer via IP and IVroute, respectively. Thus, αFR CAR BBz specific T cells inhibit tumorgrowth and ascites formation in SKOV3 murine model of peritonealcarcinomatosis. Importantly, αFR CAR BBz specific T cells improve thesurvival time.

Example 9: Lung Metastatic Model of Human Ovarian Cancer

Occasionally, ovarian cancer patients present with aggressive disease,manifested by parenchymal liver or lung metastases, or developmetastases to such distant sites as the brain during diseaseprogression. For the generation of a lung metastatic model of ovariancancer, 8-12-week-old NOG mice were i.v. injected with 2×10⁶ SKOV3 Luccells (in 200 μl PBS) on day 0. After evidence of tumor establishment inthe lungs on day 3, animals were treated with tail-vein injections of15×10⁶ either αFR CAR BBz T cells or CD19 CAR BBz T cells on day 3 andday 8 (in 200 μPBS). This route tumor inoculation establishedprogressive lung metastases in 100% of mice, as judged bybioluminescence imaging (FIG. 16 Top panel). In CD19 CAR T cells treatedmice, tumors progressively grew in all animals. In contrast, injectionsof αFR CAR BBz-specific T cells resulted in rapid regression of lungmetastasis in all treated animals (n=5) (FIG. 16 day 14). 80% (4/5) ofmice had no evidence of tumor recurrence after >2 months of follow-up.One animal had recurrent lung metastasis and was euthanized on day 70.Thus, adoptive transfer of αFR-specific T cells offers the possibilityof regression of lung metastases.

Example 10: Relevance to Human Application

The above studies demonstrate the functionality of the constructs invivo and in vitro. The multiple human ovarian cancer cell lines such asSKOV3, A1847, OVCAR3, C30, A2780 and PEO-1 were used in vitro experiment(see FIG. 9C). This approach was also in a preclinical xenograft modelusing human ovarian cancer cell line SKOV3 Luc. A possible differencebetween the in vitro system and the proposed human treatment is that thein vitro experiments were done on cell lines, which may not reflect thetumor cells in vivo.

The ovarian cancer model used is an orthotopic human xenograft and thussimilar to the human ovarian cancer disease that spreadsintraperitoneally. A possible difference with human disease is that thismodel is extremely aggressive. Another obvious difference is that themodel is developed in an immunodeficient host, while human ovariancancer develops in immunocompetent individuals, although patients withovarian cancer may exhibit elements of suppression of cellular immunity.

Example 11: Minimal Level of Gene Transfer and/or Expression that isEstimated to be Necessary for the Gene Transfer Protocol to beSuccessful in Humans and Determination of Minimal Level

A copy number release specification of 0.2-5 for the final cellularproduct has been achieved. This specification is above what has beenroutinely achieved with lentiviral vectors, and the number represents arange that is expected to demonstrate activity while minimizingunnecessary risk from extensive insertions. For Stratum 1, a singleinfusion of 3×10⁷ CAR T cells/m² I.T. can be administered as the lowestdose where as for Stratum 2, CAR T cells dosed by I.V. infusion using a“split dose” with 3×10⁸ CAR T cells/m² as the lowest dose. Finalproducts can be tested for percent transduction by flow cytometry, andthe numbers of transduced cells can be recorded after harvest and atbaseline, immediately after infusion.

A major goal of the clinical trial is to establish an optimal biologicdose for the αFR-CAR T cells. The purpose of this pilot trial is toevaluate the safety, tolerability and differential survival andtrafficking of cells modified with the αFR-CAR. The numbers of patientsproposed can be sufficient to reach this endpoint, as supported by thestatistical analysis plan in the protocol.

Example 12: Effectiveness of the Delivery System in Achieving theMinimally Required Level of Gene Transfer and Expression

For data demonstrating the in vitro efficiency of transduction of theαFR constructs, please refer to FIGS. 8A-8B, for animal data pleaserefer to FIGS. 11A-11B, 12A-12D, 13, and 14A-14C. Efficient transductionof CD3/28 stimulated autologous T cells using lentiviral vectortechnology at clinical scale using a lentiviral vector in a pilotclinical trial has been demonstrated. A similar method can be used forthe proposed study.

Example 13: Gene-Specific Expression

The lentiviral vector used for this study only encodes a single protein,which is the transgene of interest. Therefore, no other genes areexpressed by the vector other than the transgene. In previous lentiviralvector study, a conditionally replicating viral vector, which used thenative LTRs as promoters was used. There is very low basal activity inthese promoters in the absence of tat, and therefore little read throughinto neighboring genes was expected, except in the context of HIVinfection. An analysis on insertion site patterns in the five patientstreated was performed on the original transduced cellular product. Itwas found that the vector inserted into genes in a pattern similar tothat observed with other lentiviral vectors, including SIN vectors. Thelocation of insertion was as expected for lentiviruses, which ispredominantly in gene-rich regions (FIG. 3b of Levine et al, 2006). Thevector inserted into transcriptionally active genes distributedthroughout the coding region. These findings were confirmed in a morerecent publication evaluating the insertion site patterns longitudinallyin patients who received lentiviral vector transduced T cells. Noselection for integration sites was detected indicating that there is nofunctional modification of onco- or tumor suppressor genes.

The vector used in the proposed study can be a SIN vector with aconstitutively active internal promoter as described in FIGS. 6 and 7. Arecent study evaluated transcriptional activity of SIN vectors and theireffect on activation of oncogenes, in direct comparison to a murineretroviral vector with intact LTRs, in the context of a tumor pronemouse model. Molecular analysis of the genes in tumors developing in themice did not support oncogene transcriptional activation by the SINvectors, although the murine retroviral vectors induced oncogeneactivation in myeloid tumor subsets. This is a result of a greaterenhancer effect from the MLV LTR, and the insertion pattern of the MLVvector, which dominates in the 5′ control region of genes where it ismore likely to modulate gene expression.

Although transcriptional activation of neighboring genes is possible incells transduced with SIN lentiviral vectors, the frequency of the eventappears to be much lower than with MLV vectors. It is worthwhile to notethat the natural experiment has been conducted in HIV infection, where Tcell leukemia is not a known side effect of infection with the wild typelentivirus.

Example 14: Cell Expression of DNA Insert and Percentage of NormalActivity

Expression of the transgene is under the control of the ubiquitousmammalian ef-1α promoter which has previously been shown to have optimaltransgene expression in T cells, and therefore expression in alltransduced cells was observed.

The transduced T-cells ready for infusion are biologically active. Inpreclinical data in NOG mice, expression of αFR constructs in transducedcells persisted in the mouse models till day 74 (see FIG. 13). Inpatients, persistence of transduced T cells by monitoring peripheralblood by both PCR and flow cytometry can be tested throughout the trial.

Example 15: Gene Mis-Expression

An advantage of the ex vivo manufacturing process is that the cellsexposed to the vector can be carefully controlled. As a result, the αFRCAR constructs can only be expressed in cells targeted by the vectorduring cell processing. Since the T lymphocytes are isolated by negativeselection, a small percentage of monocytes and B cells can be present inthe culture during transduction. Monocytes are not present in the finalproduct but a small percentage of B cells may remain (<2%).

In the first lentiviral vector trial, the phenotype of the final productaveraged 93.4% (range 80 to >99%) CD3+ T cells. A similar targetpopulation in the present protocol is expected.

Example 16: Production of Retroviral Particles

293 cells and primary T cells have been transduced with the lentiviralvector preparation. The vector is a third generation self inactivating(SIN) vector and does not contain any viral proteins and is replicationincompetent. Therefore, no infectious particles are produced by cellsthat have been transduced with the vector.

Vector is produced in 293T cells by transient transfection. The releasetest for the lentiviral vector preparation includes a sensitivebiological assay for a replication competent lentivirus (RCL). Althoughthe chances of generation of an RCL are negligible due to the lack ofaccessory proteins and homology regions in the production system, thisfinal biological assay provides functional evidence to the lack of anyreplicating moiety in the vector preparation.

To date, no RCL has been detected in any GMP vector lots from prior andongoing clinical trials. More importantly, there is no evidence of RCLin any of the 23 patients that have been treated so far with lentiviralvector modified cells. Development lots and GMP lots of the vectors forthe proposed study are in progress and can be tested for RCL inaccordance with FDA Guidelines.

For the previous clinical study using a conditionally replicating viralvector, the stability of the vector in primary CD4 T cells was examinedand it was demonstrated that the vector copy number is stable for theduration of the experiment (FIG. 17). If significant rearrangement ofthe vector had occurred, then this would have been reflected in changesin the vector copy number. It is important to note that the vector copynumber in the experiment remained stable even though the cells expandedover 1000-fold, since these experiments showed no decrease in vectorcopy number, as measured by TaqMan PCR (sensitivity of 1 copy perreaction that contains 10,000 to 30,000 cells).

A stability study for this vector was also carried out in primary Tcells to evaluate the stability of the vector from production totransduction (i.e. a single reverse transcription step). Sequencing ofthe entire vector genome showed 100% fidelity between the productionvector and the provirus in the transduced cells. Although the error ratefor reverse transcription is about 1 in 10,000, resulting inapproximately 1 error on average for every 2 proviruses, when atransduced cellular population is taken together, such mutations likelyfall into the background as noise, thus providing an overall picture of100% fidelity. Since there is only a single round of reversetranscription for each vector, the effect of the mutation rate on thefunction of the transcript should be negligible. In order to minimizeinstability of the vector, the αFR-CAR vectors have been designed suchthat they have no direct or inverted repeat elements.

No information exists as to whether these vectors could recombine withan endogenous retro-element present in human cells. However,considerable knowledge exists about the parental wt-HIV genome and howit replicates in human cells. To date there has been no description of aproductive recombination event between wt-HIV and an endogenousretroviral element, even though 40 million humans are infected with thevirus. Given the occurrence of this hypothetical recombination event,the issue becomes whether it would result in the generation of arecombinant with greater pathogenicity than wt-HIV. If this occurs, thenit would likely have already occurred in the infected population. Thefact that no such event has been described in the worldwide infectedpopulation suggests that if a recombination event between wt-HIV and anendogenous sequence occurs, then the resulting recombinant is rare or isnot pathogenic in humans.

Example 17: Laboratory Evidence Concerning Potential Harmful Effects ofthe Transfer (e.g., Development of Neoplasia, Harmful Mutations,Regeneration of Infectious Particles, or Immune Responses)

To date there are no reports of tumorigenesis caused by an HIV-derivedlentiviral vector. A recent report by Montini et al (Montini et al.,2006) provides evidence supporting the relative biosafety of SINlentiviral vectors in terms of genotoxicity. Although it remainstheoretically possible that HIV-derived lentiviral vectors could causeinsertional oncogenesis, to date no data exist to support this theory.

The vector used in the proposed studies is a SIN vector, which has beenmeticulously designed to contain only the minimal genetic elementsrequired for function, and no vector proteins for maximum Biosafety(Dull et al., 1998). Vector is manufactured and purified under goodmanufacturing conditions, in a manner consistent with FDA requirementsfor purity as specified in the 2006 FDA Guidance on RCR testing, the USPharmacopeia Guidance on Cell and Gene Therapy Products, and the CFRregulations on Pharmaceutical and Bulk Chemical GMPs.

In the present application, the vector is used ex vivo and therefore isless likely to induce an immune response. However, in the ongoing PhaseI/II clinical trial, it was observed that generation of antibodies tothe vector envelope protein in 50% of patients after the third dose ofvector modified cells. Since the dosing schedule is biweekly, it ispossible in some or all patients that the immune response was generatedas early as by the second infusion. It was not believed that a singleinfusion would generate an antibody response since none of the patientsin the single dose Phase I clinical study became positive for VSV-G Ab(Levine et al., 2006). Generation of antibody to VSV-G did not impactthe persistence of vector modified cells in the patients, nor did itcoincide with adverse events in the patients. Therefore, the developmentof VSV-G antibodies has little to no detectable clinical impact. In theproposed clinical study, two infusions per patient are given. Therefore,it is possible to observe an antibody response to VSV-G in this trial.

Example 18: Animal Studies for Pathogenicity

Extensive biodistribution and biotoxicity studies were performed for thefirst lentiviral vector clinical trial. For the biodistribution study, atotal of 192 mice were divided into four groups of 48 mice who receiveda 0.3 ml tail vein injection of the following: infusion media alone(group 1), 20 million mock transduced human T cells (group 2), 300,000vector transduced human T cells (group 3) or 20 million vectortransduced human T cells (group 4). Male and female mice were testedseparately. Mice were analyzed at days 2, 15, 30, 91, and 123, and micewere evaluated for in-life parameters which included mortality, clinicalobservations/physical examinations, and body weights. The study showedthat vector transduced human T cells had no effect on mortality,clinical observations or body weights. No mice developed T cell tumors.The presence of vector sequences was assessed by PCR. On days 2 and 30,vector sequences were present in all tissues tested from groups 3 and 4.By day 91, only lung, liver, spleen and tail from four mice werepositive for vector, and by day 123 no vector was detected (Table 1).

TABLE 1 Results from a biodistribution study evaluating lentiviralvector- transduced human T cells in immunodeficient mice. Shown are theresults from the mice given the highest dose of transduced cells (20million human CD4 cells per mouse i.v.) as described in the text. Maleand female mice are combined for n = 10 per timepoint. Tissue Day 2 Day30 Day 91 Day 123 Heart 100% 30% * * Gonads  80% 50% * *(testes/ovaries) Liver 100% 90% 10% * Inguinal lymph  90% 20% * * nodeBone Marrow 100% * * * Lung 100% 40% * * Spleen 100% 10% 10% * Tail 100%40% 10% * Blood 100% 40% * * * no vector detected.

In other experiments, the biotoxicity of lentiviral vector modified Tcells was evaluated a total of 144 mice divided into four groups of 36mice per group who received a tail vein injection of 0.3 ml divided intothe same groups as listed above. Male and female mice were testedseparately. Mice were evaluated for mortality, clinicalobservations/physical examinations, body weights, clinical pathologyresults, gross pathology findings, organ weight data, bone marrowevaluation, and histopathology results. The results from this study werethat vector transduced human T cells were not associated with anytoxicity, and had no effect on any of the parameters evaluated.

The vector used for the proposed study is different in structure andpayload, however the lentiviral transduced cellular vehicle (human Tcells) is anticipated to behave in a similar manner in the mouse model.

The genotoxicity of SIN vectors has recently been tested in a tumorprone mouse model, and no toxicity could be detected within thesensitivity of the model, although toxicity with a murine retrovirusvector was detected. Studies for tolerability of the αFR-CAR cells canonly be accurately determined in an early phase clinical trial, as evenin the humanized mouse model, antigen, T cell trafficking, and tumorantigen expression across various tissues can be different in the animalmodel.

There is no evidence to support vector DNA mobilizing or enteringuntreated cells. In the biodistribution study described above, thevector sequence was only detected in vivo in mice in conjunction withhuman cell markers, indicating that it did not mobilized to non-targetedcells. Gonads in mice did not have detectable vector sequences at days91 or 123 (end of study) (Table 1). This can be evaluated as well in theplanned biotoxicity and biodistribution study for the αFR CAR.

All preclinical animal studies have been conducted in theimmunodeficient NOD/SCIDγ−/− (NSG) human xenotransplantation animalmodel.

Example 19: In Vivo Persistence, Tumor Localization, and AntitumorActivity of CAR-Engineered T Cells is Enhanced by CostimulatorySignaling Through CD137 (4-1BB)

Human T cells engineered to express a chimeric antigen receptor (CAR)specific for folate receptor-α (FRα) have shown robust antitumoractivity against epithelial cancers in vitro but not in the clinicbecause of their inability to persist and home to tumor in vivo. In thisstudy, CARs were constructed containing a FRα-specific scFv (MOv19)coupled to the T-cell receptor CD3ζ chain signaling module alone(MOv19-ζ) or in combination with the CD137 (4-1BB) costimulatory motifin tandem (MOv19-BBζ). Primary human T cells transduced to expressconventional MOv19-ζ or costimulated MOv19-BBζ CARs secreted variousproinflammatory cytokines, and exerted cytotoxic function whenco-cultured with FRα⁺ tumor cells in vitro. However, only transfer ofhuman T cells expressing the costimulated MOv19-BBζ CAR mediated tumorregression in immunodeficient mice bearing large, established FRα+ humancancer. MOv19-BBζ CAR T-cell infusion mediated tumor regression inmodels of metastatic intraperitoneal, subcutaneous, and lung-involvedhuman ovarian cancer. Importantly, tumor response was associated withthe selective survival and tumor localization of human T cells in vivoand was only observed in mice receiving costimulated MOv19-BBζ CAR Tcells. T-cell persistence and antitumor activity were primarilyantigen-driven; however, antigen-independent CD137 signaling by CARimproved T-cell persistence but not antitumor activity in vivo. Resultsdescribed herein show that anti-FRα CAR outfitted with CD137costimulatory signaling in tandem overcome issues of T-cell persistenceand tumor localization that limit the conventional FRα T-cell targetingstrategy to provide potent antitumor activity in vivo.

As described herein, the issue of limited FRα-specific T-cellpersistence and tumor activity in vivo is addressed through theintroduction of the CD137 costimulatory signaling domain into aFRα-specific CAR and studied the role of CD137 signaling in FRα-directedCAR T-cell therapy of human cancer. Compared with “first-generation” CARthat provide CD3ζ signaling to T cells but lack cis costimulatorysignaling capacity, T cells expressing FRα-specific CAR with a CD137signaling domain in tandem showed minimally improved antitumor activityin vitro, but markedly superior tumor regression capacity in establishedhuman ovarian cancer xenograft models, which was associated withenhanced T-cell persistence and tumor localization in vivo. Tumorregression and T-cell persistence were both attainable by various routesof T-cell infusion, and intravenous (i.v.) cell infusion mediates theregression of human cancer in xenograft models of advancedintraperitoneal (i.p.), subcutaneous (s.c.), and lung-involvedmetastatic disease. T-cell persistence and tumor activity in vivo werelargely antigen-driven; however, provision of CD137 signaling in theabsence of specific antigen recognition by CAR could improve T-cellpersistence but not antitumor activity in vivo. Incorporation of theCD137 signaling domain in FRα-specific CARs thus overcomes thelimitation of past CAR approaches by improving the persistence oftransferred T cells in vivo, and bolstering their accumulation in tumorand antitumor potency.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Anti-FRα Chimeric Immune Receptor Construction

The chimeric immune receptor backbone constructs were generated aspreviously described (Carpenito et al., 2009, Proc Natl Acad Sci USA106:3360-65). The anti-FRα scFv sequence was derived from MOv19 (Miottiet al., 1987, Int J Cancer 39: 297-303; Figini et al., 1998, Cancer Res58(5):991-6), a monoclonal antibody directed against FRα. The MOv19 scFvhas been fully characterized (Figini et al., 2009, Cancer ImmunolImmunother 58(4):531-46; Melani et al., 1998, Cancer Res 58: 4146-54)and was amplified using the following primers:

(SEQ ID NO: 24) 5′-GCGGGATCCTCTAGAGCGGCCCAGCCGGCCATGGCCCAGGTG-3′(Bam-HI is underlined) and (SEQ ID NO: 25)5′-GCGGCTAGCGGCCGCCCGTTTTATTTCCAACTTTGTCCCCCC-3′ (Nhe-I is underlined)and then cloned into the CAR backbone vector. The scFv PCR product wasdigested with BamHI and NheI endonucleases and gel purified beforeligation into the pCLPS vector, a third generation self-inactivating CMVpromoter based lentiviral expression vector based onpRRL-SIN-CMV-eGFP-WPRE (Dull et al., 1998, J Virol 72(11):8463-71). Theanti-CD19-BBζ CAR construct has been previously described (Milone etal., 2009, Mol Ther 17:1453-64). High-titer lentiviral vectors wereproduced and concentrated 10-fold by ultracentrifugation for 3 h at26,000 rpm as previously described (Parry et al., 2003, J Immunol171:166-74).

Cell Lines

Lentivirus packaging was performed in the immortalized normal fetalrenal 293T cell line purchased from ATCC. Human cell lines used inimmune based assays include the established human ovarian cancer celllines SKOV3, A1847, OVCAR3, C30, and PEO-1. For bioluminescence assays,target cancer cell lines were transfected to express firefly luciferase(fLuc), enriched by antibiotic selection positive expression bybioluminescence imaging. For specificity controls, the mouse malignantmesothelioma cell line, AE17 was transduced with lentivirus to expressFRα (AE17.FRα). CD19-expressing K562 (CD19+K562) cells, a humanerythroleukemic cell line, were obtained (Milone et al., 2009, Mol Ther17:1453-64). 293T cells and tumor cell lines were maintained inRPMI-1640 (Invitrogen) supplemented with 10% (v/v) heat-inactivated FBS,2 mM L-glutamine, 100 g/mL penicillin and 100 U/mL streptomycin. Allcell lines were routinely tested for mycoplasma contamination.

Human T Cells

Primary human CD4+ and CD8+ T cells, which were purchased from the HumanImmunology Core at University of Pennsylvania, were isolated fromhealthy volunteer donors following leukapheresis by negative selection.All specimens were collected under a protocol approved by a UniversityInstitutional Review Board, and written informed consent was obtainedfrom each donor. T cells were cultured in complete media (RPMI 1640supplemented with 10% heat inactivated FBS, 100 U/mL penicillin, 100ρμg/mL streptomycin sulfate, 10 mmol/L HEPES), and stimulated withanti-CD3 and anti-CD28 monoclonal antibodies (mAb)-coated beads(Invitrogen) as described (Levine et al., 1997, J Immunol 159:5921-30).Twelve to twenty-four hours after activation, T cells were transducedwith lentiviral vectors at multiplicity of infection of approximately 5to 10. CD4+ and CD8+ T cells used for in vivo experiments were mixed at1:1 ratio, activated, and transduced. Human recombinant interleukin-2(IL-2; Novartis) was added every other day to a 50 IU/mL finalconcentration and a cell density of 0.5×10⁶ to 1×10⁶ cells/mL wasmaintained. Once T cells seemed to rest down, as determined by bothdecreased growth kinetics and cell sizing by using the Multisizer 3Coulter Counter (Beckman Coulter), engineered T-cell cultures wereadjusted to equalize the frequency of transgene expressing cells priorto functional assays.

Flow Cytometric Analysis

The following MAbs were used for phenotypic analysis: APC-Cy7 MouseAnti-Human CD3; FITC anti-human CD4; APC anti-human CD8; PE-anti humanCD45. All mAbs were purchased from BD Biosciences PharMingen. In T celltransfer experiments, peripheral blood was obtained via retro-orbitalbleeding and stained for the presence of human CD45, CD4, and CD8 Tcells. After gating on the human CD45+ population, the CD4+ and CD8+subsets were quantified using TruCount tubes (BD Biosciences) with knownnumbers of fluorescent beads as described in the manufacturer'sinstructions. Tumor cell surface expression of FRα was detected byMov18/ZEL antibody (Enzo Life Sciences). FRα specific CAR expression wasdetected by PE conjugated goat anti-mouse IgG F(ab′)₂ (specific forscFvs of murine origin) that was purchased from Jackson ImmunoResearch.For intracellular staining, cells were fixed, permeabilized, and stainedwith PE-conjugated anti-Bcl-XL antibody (Southern Biotech). Isotypematched control Abs were used in all analyses. Flow cytometric data wereanalyzed by FlowJo software.

Cytokine Release Assays

Cytokine release assays were performed by co-culture of 1×10⁵ T cellswith 1×10⁵ target cells per well in triplicate in 96-well round bottomplates in a final volume of 200 μl of T cell media. After 20-24 hr,co-culture supernatants were assayed for presence of IFN-γ using anELISA Kit, according to manufacturer's instructions (Biolegend). Valuesrepresent the mean of triplicate wells. IL-2, IL-4, IL-10, TNF-αcytokines were measured by flow cytometry using Cytokine Bead Array,according to manufacturer's instructions (BD Biosciences).

Cytotoxicity Assays

For the cell based bioluminescence assay, 5×10⁴ firefly Luciferaseexpressing (fLuc⁺) tumor cells were cultured with complete media in thepresence of different ratios of transduced T cells using a 96-wellMicroplate (BD Biosciences). After incubation for 18-20 hours at 37° C.,each well was filled with 50 μl DPBS resuspended with 1 μl D-luciferin(0.015 g/ml) and imaged using a Xenogen IVIS Spectrum. Percent tumorcell viability was calculated as the mean luminescence of theexperimental sample minus background divided by the mean luminescence ofthe input number of target cells used in the assay minus backgroundtimes 100. All data are represented as a mean of triplicate wells. ⁵¹Crrelease assays were performed as described (Johnson et al., 2006, JImmunol 177(9):6548-59). Target cells were labeled with 100 μCi 51Cr at37° C. for 1.5 hours. Target cells were washed three times in PBS,resuspended in CM at 10⁵ viable cells/mL and 100 μL added per well of a96-well V-bottom plate. Effector cells were washed twice in CM and addedto wells at the given ratios. Plates were quickly centrifuged to settlecells, and incubated at 37° C. in a 5% CO₂ incubator for 4 or 8 hoursafter which time the supernatants were harvested and counted using a1450 Microbeta Liquid Scintillation Counter (Perkin-Elmer). Percentspecific lysis was calculated as (experimental−spontaneouslysis/maximal−spontaneous lysis) times 100. For gfp target cell lysisassays, transduced T cells were co-cultured at various effector totarget ratios for 24 hrs with 5×10⁴ gfp expressing AE17 or AE17.FRαcells and photographed under fluorescent microscopy. Target cell lysiswas indicated by imaging reduction in gfp-labeled adherent tumor cells.

Xenograft Model of Ovarian Cancer

Mouse studies were carried out as previously described (Carpenito etal., 2009, Proc Natl Acad Sci USA 106:3360-5, Milone et al., 2009, MolTher 17:1453-64) with modifications detailed herein. All animals wereobtained from the Stem Cell and Xenograft Core of the Abramson CancerCenter, University of Pennsylvania. Eight to 12-weekoldNOD/SCID/γ-chain−/−(NSG) mice were bred, treated and maintained underpathogen-free conditions in-house under University of Pennsylvania IACUCapproved protocols. For an established ovarian cancer model, 6 to12-week-old female NSG mice were inoculated s.c. with 3×10⁶ SKOV3 fLuc+cells on the flank on day 0. After tumors become palpable at about 1month, human primary T cell (CD4+ and CD8+ T cells used were mixed at1:1 ratio) were activated, and transduced as described elsewhere herein.After 2 weeks T cell expansion, when the tumor burden was ˜200-300 mm³,mice were treated with T cells. The route, dose, and timing of T-cellinjections are indicated elsewhere herein. Tumor dimensions weremeasured with calipers, and tumor volumes calculated using the formulaV=1/2(length×width²), where length is greatest longitudinal diameter andwidth is greatest transverse diameter. Animals were imaged prior to Tcell transfer and about every week thereafter to evaluate tumor growth.Photon emission from fLuc+ cells was quantified using the “Living Image”software (Xenogen) for all in vivo experiments. Tumors were resectedimmediately after euthanasia approximately 40 days after first T celldose for size measurement and immunohistochemistry. For theintraperitoneal model of ovarian cancer, 8 to 12-week-old NSG mice wereinjected i.p. with 5×10⁶ SKOV3 fLuc+ cells. Thirty days after peritonealinoculation, mice bearing well-established SKOV3 tumors were dividedinto groups and treated. Mice were sacrificed and necropsied when themice became distressed and moribund. Lung metastases were established byinjecting 2×10⁶ SKOV3 fLuc+ cells into the tail vein of female NSG mice.After evidence of tumor establishment in the lungs on day 3, animalswere treated with tail-vein injections of engineered T cells on day 3and day 8. To monitor the extent of tumor progression, the mice wereimaged weekly or biweekly and body weights of the mice were measured. Inall models, 4-5 mice were randomized per group prior to treatment.

Bioluminescence Imaging

Tumor growth was also monitored by Bioluminescent imaging (BLI). BLI wasdone using Xenogen IVIS imaging system and the photons emitted fromfLuc-expressing cells within the animal body were quantified usingLiving Image software (Xenogen). Briefly, mice bearing SKOV3 fLuc⁺ tumorcells were injected intraperitoneally with D-luciferin (150 mg/kg stock,100 μL of D-luciferin per 10 grams of mouse body weight) suspended inPBS and imaged under isoflurane anesthesia after 5-10 minutes. Apseudocolor image representing light intensity (blue, least intense;red, most intense) was generated using Living Image. BLI findings wereconfirmed at necropsy.

Immunohistochemistry

Mice were euthanized by CO₂ inhalation and tumors were collected inTissue-Tek O.C.T. Compound, and frozen at −80° C. A standardStrept-avidin horseradish immunoperoxidase method was used for human CD3staining. Primary and secondary antibodies were diluted in buffercontaining 10% normal goat serum. 7 m cryosections were fixed in coldacetone for 5 min at 4° C. and blocked with Dako's (Carpentaria, CA)peroxidase blocking system for 10 minutes. Sequential incubationsincluded the following: 10% normal goat serum (30 min at roomtemperature (RT)); primary rabbit anti-human CD3 monoclonal antibody(Thermo Scientific RM-9107) at 1:100 dilution (45 min. at RT); secondarybiotinylated goat anti-rabbit antibody at 1:200 dilution (30 min at RT);strept-avidin-biotinylated horseradish peroxidase complex reagent (Dako)(30 min at RT); and three 5 minute washes in buffer after eachincubations. Sections were then exposed to the chromagen DAB plus fromDako for 5 min at RT and counterstained with hematoxylin, dehydrated,cleared and mounted.

Statistical Analysis

Statistical analysis was carried out by 2-way repeated measures ANOVAfor the tumor burden (tumor volume, photon counts). Student's t test wasused to evaluate differences in absolute number of transferred T cells,cytokine secretion, and specific cytolysis. Kaplan-Meier survival curveswere compared by using the log-rank test. GraphPad Prism 4.0 (GraphPadSoftware) was used for the statistical calculations. P<0.05 wasconsidered significant.

The results of the experiments are now described.

CAR Construction

The mouse anti-human FRα-specific scFv MOv19 was selected on the basisof its high binding affinity for FRα (10⁸-10⁹ M⁻¹; refs. Miotti et al.,1987, Int J Cancer 39:297-303; Melani et al., 1998, Cancer Res58:4146-54; Figini et al., 1998, Cancer Res 58:991-6). FRα CARconstructs were comprised of the MOv19 scFv linked to a CD8α hinge andtransmembrane region, followed by a CD3ζ signaling moiety alone(MOv19-ζ) or in tandem with the CD137 intracellular signaling motif(MOv19-BBζ; FIG. 18A). A signaling deficient FRα-specific CAR containinga truncated CD3ζ intracellular domain (MOv19-Δζ) was designed to assessthe contribution of CD3ζ signaling. An anti-CD19 CAR containing CD3ζ andCD137 signaling motifs in tandem (anti-CD19-BBζ) was used as an antigenspecificity control (Milone et al., 2009, Mol Ther 17:1453-64). CARconstructs were subcloned into the pCLPS lentiviral vector wheretransgene expression is driven off the cytomegalovirus promoter. Usinggene transfer technology established for clinical application,lentiviral vectors efficiently transduced primary human T cells toexpress the anti-FRα CAR (FIG. 18B). T-cell transduction efficiency, asassessed by flow cytometry, was equilibrated for all constructs atapproximately 50% in all assays.

Primary Human FRα CAR T Cells Exert Antigen-Specific Function In Vitro

Because ovarian cancer frequently express FRα (Miotti et al., 1987, IntJ Cancer 39:297-303), a panel of established human ovarian cancer celllines that express surface FRα at varying levels (SKOV3, A1847, andOVCAR3) was selected for assays (FIG. 18C). Two ovarian cancer lines,C30 and PEO-1, were negative for FRα. Transduced T cells expressingMOv19-BBζ or MOv19-ζ CARs recognized FRα ⁺ tumor lines and secreted highlevels of IFN-γ, but not when stimulated with FRα-lines (FIG. 18D).FRα-specific CAR T cells also secreted high levels of IL-2 and TNF-αwhen stimulated with FRα cancer cells and low but detectable levels ofIL-4 and IL-10 (FIG. 24). MOv19 CARs functioned in both primary humanCD4+ and CD8+ T cells. In all cases, MOv19-BBζ T cells secreted moreIFN-γ than MOv19-ζ T cells after specific stimulation. CD19-BBζ CAR didnot produce IFN-γ, except when co-incubated with K562 cells engineeredto express surface CD19 antigen, and human T cells expressingMOv19-ΔζCAR did not secrete cytokine when stimulated with FRα F cancercells (FIG. 18D), showing that antigen specificity and CD3ζ signalingare required for CAR activity in T cells.

To interrogate antigen-specific cytolytic potential, anti-FRα CAR CD8+ Tcells were co-cultured with FRα-AE17 (Jackaman et al., 2003, J Immunol171:5051-63), a mouse malignant mesothelioma cell line, or AE17.FRα (anAE17 line derivative transduced to express high surface levels of humanFRα). In standard 4-hour chromium release and 24-hour bioluminescenceassays, FRα-specific CAR T cells (MOv19-ζ and MOv19-BBζ) specificallylysed AE17.FRα cells but not the parental AE17 line (FIGS. 25A-25C). Tcells expressing anti-CD19-BBζ, MOv19-Δζ, or green fluorescent protein(gfp) did not lyse AE17.FRα or AE17 cells. Consistent with cytokineproduction results, primary human CD8⁺ T cells expressing MOv19-ζ orMOv19-BBζ CAR directly and efficiently lysed FRα+ human ovarian cancercell lines SKOV3 and A1847, but not FRα-lines C30 or 624mel, a melanomacell line (FIG. 18E). MOv19-BBζ CAR T cells exhibited increasedcytotoxicity compared with MOv19-ζ CAR T cells, but not at a level ofstatistical significance. Thus, human T cells transduced withFRα-specific CAR specifically recognize FRα+ human and mouse cancercells and exert MHC-unrestricted cytotoxic activity in vitro.

Antitumor Activity of Primary Human FRα CAR T Cells In Vivo

CAR functional activity in vitro cannot adequately predict the antitumorpotential of transduced human T cells in vivo. The antitumor efficacy ofFRα CAR constructs were evaluated in a xenograft model of large,established cancer. Immunodeficient NOD/SCID/IL-2Rγc^(null) (NSG) micewere inoculated s.c. with firefly luciferase (fLuc)-transfected FRα+SKOV3 human ovarian cancer cells on the flank and received intratumoral(i.t.) injections of CAR+ T cells on days 40 and 45 post-tumorinoculation (p.i.), when tumors were 250 mm³ or more in size. Tumors inmice receiving saline, MOv19-ΔζCAR T cells, or gfp T cells progressedbeyond the time of T cell transfer as measured by caliper-based sizingand bioluminescence imaging (BLI; FIG. 19A and FIG. 19B). Tumor growthwas modestly delayed in mice receiving MOv19-ζ T cells (P=0.027),compared with all 3 control groups at the latest evaluated time point(38 days after first T-cell dose). In contrast, mice receiving i.t.injection of MOv19-BBζ T cells experienced rapid tumor regression, whichwas significantly better than MOv19-ζ T cells (P<0.001), indicating thatincorporation of CD137 signals enhances overall antitumor activity invivo. Tumor-bearing mice treated with MOv19-BBζ-transduced T cellsdelivered via i.v., i.p. injection, or i.t. routes experienced tumorregression (FIG. 19C). Following i.v. or i.p. infusion of MOv19-BBζ Tcells, antitumor activity was again observed, though delayed inregression by approximately 7 days relative to i.t. delivery, indicatingthat although local injection is optimal, systemically infused CAR Tcells can marginalize upon adoptive transfer to mediate potent antitumoreffects in vivo.

Persistence of Primary Human FRα CAR T Cells In Vivo is Increased by4-1BB Signals

Without wishing to be bound by any particular theory, it is believedthat the persistence of transferred tumor-reactive T cells followingadoptive T-cell therapy is highly correlated with tumor regression(Robbins et al., 2004, J Immunol 173:7125-30). In experiments describedelsewhere herein, peripheral blood was collected from tumor-bearing mice3 weeks after the last T-cell dose and quantified for persistent humanCD4⁺ and CD8⁺ T cells (FIG. 19D). CD4⁺ and CD8⁺ T-cell counts werehighest in mice receiving MOv19-BBζ CAR T cells, whether delivered byi.t., i.p., or i.v. routes of administration, compared with gfp,MOv19-Δζ, and MOv19-ζ treatment groups. Notably, human T-cell counts inmice receiving MOv19-BBζ CAR T cells by i.v. injection was significantlyhigher than those in the parallel MOv19-ζ CAR group (P<0.01), indicatinga role for CD137 in T-cell survival in vivo. There was no significantdifference in level of T-cell persistence among mice receiving MOv19-BBζCAR T cells by i.v., i.t., or i.p. injection (P=0.2), despite a trendtoward less cells in the i.v. injection group. Total T-cell counts inthe MOv19-ζ treatment group was statistically similar to other controlgroups including mice receiving saline in the absence of human T-cellinjection (FIGS. 26A-26D; P>0.05), suggesting that antigen specificityalone is not sufficient for T-cell maintenance in vivo. This wasprimarily attributed to poor CD4+ T-cell persistence because circulatingMOv19-ζ CAR CD8+ T cells persisted at greater numbers than MOv19-ΔζCAR(P=0.026) or gfp (P=0.013) cells. Four weeks after last MOv19-BBζ CART-cell dose, the absolute number of human T cells persisting in theblood was inversely correlated with tumor burden of each group (FIGS.26A-26D; r=−0.78). Tumor BLI results were consistent with the size ofresected residual tumors (FIG. 27). While not wishing to be bound by anytheory, enhanced persistence of MOv19-BBζ CAR T cells, compared withMOv19-ζ, seemed to be attributed in part to an increased upregulation ofanti-apoptotic Bcl-XL protein expression after antigen stimulation(FIGS. 26A-26D). Thus, tumor regression was associated with the stablepersistence of engineered human T cells in vivo and supported byprovision of CD137 costimulation.

Tumor Regression and T-Cell Persistence are Antigen-Driven In Vivo

To determine whether MOv19-BBζ CAR antitumor activity isantigen-specific, a comparative study was conducted with ananti-CD19-specific CAR also containing the CD137 signaling domain(Milone et al., 2009, Mol Ther 17:1453-64). NSG mice with establisheds.c. SKOV3 fLuc⁺ tumor receiving 2 i.t. T-cell injections experiencedrapid tumor regression, whereas tumor grew progressively in mice treatedwith T cells expressing gfp or CD19-BBζ CAR (FIG. 20A), excludingalloreactivity as a mechanism of tumor regression. Mice receivingMOv19-BBζ T cells had significantly higher human CD4+ and CD8+ T cellcounts than mice in anti-CD19 CAR or gfp groups (FIG. 20B; P=0.009),indicating that tumor antigen recognition drives the survival of theadoptively transferred T cells in vivo. Interestingly, T-cellpersistence was reproducibly higher in mice receiving anti-CD19-BBζ CART cells than gfp T cells (P=0.012), suggesting that persistence of CAR Tcells can be promoted in part through a CD137-driven process that doesnot require scFv engagement with antigen. Nevertheless, there was nostatistical difference in tumor control between anti-CD19-BBζ CAR andgfp groups (P=0.065) even at the latest time point studied (day 73),showing that persistence in the absence of antigen specificity isinsufficient to mediate tumor response. In this line, CAR expressingT-cell frequency in the blood of tumor-bearing mice administeredMOv19-BBζ T cells was higher than that observed in mice receivingCD19-BBζ CAR T cells, though not at statistical significance (FIG. 20C;P=0.08). However, coupled with increased T-cell counts, the total numberof circulating CAR⁺ T cells persisting 1 month after infusion weresignificantly higher in mice receiving MOv19-BBζ T cells (76±13cells/μL; P=0.013); mice in CD19-BBζ CAR and gfp groups had little to nodetectable persistence of CAR⁺ T cells with counts of 12±4 cells/μL and0±0 cells/μl, respectively (FIG. 20D). Consistent with the increasedpersistence of MOv19-BBζ T cells in the blood of treated animals,immunohistochemical analysis revealed robust accumulation of human CD3+T cells in regressing SKOV3 lesions 6 weeks after i.v. T-celladministration (FIG. 21). Few CD3⁺ T cells were detected in tumorsresected at the same time from mice that received anti-CD19-BBζ CAR orgfp-transduced T cells.

Tumor Regression in the Metastatic Disease Setting

Advanced ovarian cancer is a disease usually confined to the peritonealcavity with occasional metastatic spread to the pleural compartment. Axenogeneic model of advanced i.p. metastatic cancer was established toevaluate the functional activity of FRα-specific T cells against tumorlocalized to a more physiologically relevant compartment. NSG mice thatwere inoculated i.p. with SKOV3 fLuc⁺ cells efficiently developedperitoneal carcinomatosis which was readily evident 30 days p.i., whenMOv19-BBζ or control anti-CD19-BBζ CAR T-cell therapy was administered(FIG. 22A). Within 3 weeks of T-cell transfer, all mice that receivedcontrol anti-CD19-BBζ CAR T cells developed distended abdomens, markedbloody ascites of approximately 5 to 8 mL volume and multiple nodularperitoneal tumors, and had to be euthanized due to tumor-associated,abdominal distention (FIG. 22B and FIG. 22C). By comparison, micetreated with MOv19-BBζ CAR T cells did not develop distended abdomens orascites, and exhibited a profound enhancement in tumor-related survival(P=0.0002) with no cases of tumor-related mortality in the MOv19-BBζ CARgroup (FIG. 22C). At the time of euthanasia of mice treated withMOv19-BBζ, tumor burden was minimal to none, but mice requiredeuthanizing due to signs of distress compatible with GVHD that developsin NSG mice following xenogeneic transfer of activated human lymphocytes(King et al., 2009, Clin Exp Immunol 157:104-18). Still, median survivaltimes of 52 days after last T-cell infusion by i.v. injection and 68days by the i.p. route were observed in mice treated with MOv19-BBζ CAR,compared with 9 and 12 days in the anti-CD19-BBζ CAR T-cell groups,respectively (MOv19-BBC i.p. vs. anti-CD19-BBζ i.p., P=0.0023; MOv19-BBCi.v. vs. anti-CD19-BBC i.v., P=0.0025; FIG. 22D). Two months aftertreatment with MOv19-BBζ CAR cells via i.p. or i.v. routes, 60% (3 of 5)and 40% (2 of 5) of tumor-inoculated mice remained alive, respectively.

Occasionally, ovarian cancer patients develop lung metastases andpleural ascites formation requiring thoracentesis or other supportivemanagement procedures during disease progression (Sood et al., 1999,Clin Cancer Res 5:2485-90). A model of metastatic ovarian cancer of lungwas generated by inoculation of NSG mice with SKOV3 fLuc⁺ cells viatail-vein injection resulting in progressive lung metastases in 100% ofmice 3 days p.i. (FIGS. 23A-23B). Two i.v. injections of MOv19-BBζ Tcells resulted in rapid regression of lung metastasis in all treatedanimals 14 days p.i. and 80% (4 of 5) of mice had no evidence ofrecurrence after 1 month. By contrast, disease progression occurred inall mice receiving anti-CD19-BBζ T cells.

Tumor Response and T-Cell Persistence is Evoked by Provision of CD137Costimulatory Signals to Anti-FRα CAR T Cells

CARs combine the high affinity and specificity of antigen-specificantibody, which binds cell surface determinants in a non-MHC-restrictedmanner, with the potent effector functions of T lymphocytes (Gross etal., 1989, Proc Natl Acad Sci USA 86:10024-8). Genetically retargetingof primary human lymphocytes with CARs recognizing tumor-associatedantigens offers a robust and rapid avenue toward the generation oftumor-reactive T cells for therapy. To date, CAR-based therapy has shownpromising but often limited clinical activity, despite the reproducibledemonstration of strong effector activity in vitro (Park et al., 2007,Mol Ther 15:825-33; Pule et al., 2005, Mol Ther 12:933-41; Kochenderferet al., 2010, Blood 116:4099-102; Till et al., 2008, Blood 112:2261-71;Kershaw et al., 2006, Clin Cancer Res 12: 6106-15). Effective adoptiveT-cell therapy not only requires antitumor activity, but also in vivoexpansion and persistence of the infused tumor-reactive T cells (Robbinset al., 2004, J Immunol 173:7125-30). The experiments described hereinhave addressed the central issue of limited CAR T-cell persistence andtumor activity in vivo (Kershaw et al., 2006, Clin Cancer Res 12:6106-15) through the introduction of the CD137 (4-1BB) costimulatorysignaling domain into a Mov19 scFv-based CAR.

CD137 is a TNF receptor family member that plays an important role inT-cell proliferation and survival, particularly for T cells within thememory T-cell pool (Shuford et al., 1997, J Exp Med 186:47-55; Takahashiet al., 1999, J Immunol 162:5037-40; Suhoski et al., 2007, Mol Ther15:981-8). CD137 was selected on the basis of its demonstrated capacityto support of CD8 T-cell expansion (Suhoski et al., 2007, Mol Ther15:981-8), and upregulate important antiapoptotic protein Bcl-XLexpression (Lee et al., 2002, Eur J Immunogenet 29:449-52), and resultsshowing that adoptive transfer of tumor-specific T cells costimulated exvivo with 4-1BBL supports persistence and antitumor activity in vivo (Yiet al., 2007, Cancer Res 67:10027-37). Like the “first-generation”Mov19-ζ CAR expressing CD3ζ signaling alone, T cells engineered toexpress a “second-generation” Mov19-BBζ CAR containing CD3ζ signalingand a CD137 signaling domain in tandem preferentially secrete highlevels of Th1 cytokines including IFN-γ, TNF-α, and IL-2 upon tumorencounter and exert strong antitumor activity in vitro. Here, IFN-γcytokine production levels were generally associated with the level ofFRα expressed by tumor cell targets, and cytolysis of tumor cells byMov19-ζ CAR and Mov19-BBζ CAR T cells was efficient even at a 3:1effector to target cell (E/T) ratio in vitro. In all in vitro antitumorassays, engineered T cells expressing Mov19-BBζ CAR outperformed Mov19-ζCAR T cells, albeit not always to the level of statistical significance.Interestingly, the single exception was in the level of Th2 cytokinesecretion induced by tumor stimulation, where FRα engagement by Mov19-ζCAR T cells induced greater IL-4 and IL-10 production, suggesting thatcombined CD3ζ and CD137 signaling enforces a Th1 skewed response.

The dichotomy between first- and second-generation CAR vectors was mostevident in in vivo studies where CD137 bearing Mov19-BBζ CAR T cellsfacilitated superior regression of large vascularized tumors in anestablished human ovarian cancer xenograft model, whereas tumorprogression was almost unabated with Mov19-ζ CAR T cells. Transfer of16×10⁶ total Mov19-BBζ CAR T cells eliminated an estimated 2.5×10⁸ tumorcells (assuming that a 250 mm³ tumor mass contains approximately 2.5×10′cells); in effect, an approximately 1:15 E/T ratio. Consistent withprevious clinical observations (Dudley et al., 2002, Science 298:850-4;Robbins et al., 2008, J Immunol 180:6116-31), tumor response wasassociated with enhanced T-cell persistence and tumor localization ofMov19-BBζ CAR T cells in vivo, which, without being held to anyparticular theory, seemed to be attributed in part to upregulatedexpression of Bcl-XL following stimulation with tumor. Tumor regressionwas antigen-specific, as transfer of anti-CD19-BBζ T cells had no impacton tumor progression. Tumor regression and T-cell persistence wereattainable via systemic or local T-cell delivery, showing the capacityof transferred T cells to circulate, home to tumor and perform antitumorfunctions. Without being held to any particular theory, although i.v.injections are favorable in clinical application due to the ease ofadministration and effective in the model, data presented hereinsuggests that local administration of T cells may provide optimaltherapeutic effect, which may be in part due to increased T-celltrafficking to tumor and provision of favorable E/T ratios. However,such delivery may not be applicable for tumors with multiple grossmetastatic sites or micrometastases.

Although Mov19-BBζ and anti-CD19-BBζ T cells could be detected in theperipheral blood 3 weeks after T-cell infusion, the accumulation ofMov19-BBζ, but not anti-CD19-BBζ T cells, in FRα+ tumor lesions suggeststhat antigen-selective retention of CAR bearing T cells in tumor occursand may be requisite in part for tumor regression (Mukai et al., 1999,Cancer Res 59:5245-9). In a previous study, transferred TCR transgenic Tcells migrated indiscriminately early after adoptive transfer butexperienced antigen-dependent activation exclusively in antigen-positivetumor resulting in tumor destruction (Palmer et al., 2004, J Immunol173:7209-16). Transfer of chemokine receptor expressing CAR T cells canenforce preferential migration to tumor sites to boost antitumoractivity in vivo (Craddock et al., 2010, J Immunother 33:780-8). Resultspresented herein support the hypothesis that T-cell persistence,localization, and tumor activity in vivo are largely antigen-dependent,likely linked, processes. Notably, the use of anti-CD19-BBζ T cells asspecificity control in the assays, however, shows that provision ofCD137 signaling by CAR permitted improved T-cell persistence but notantitumor activity in vivo through a mechanism that is independent ofscFv engagement with antigen, suggestive of low-level constitutiveactivity by the CD137 module, consistent with previous data (Milone etal., 2009, Mol Ther 17:1453-64). Without being held to any particulartheory, it remains possible that persistence of nonspecificCD137-costimulated human T cells was driven by low-level TCR recognitionof xenoantigens in mice combined with constitutive CD137 signaling byCAR, as shown by the occurrence of graft-versus-host manifestations,which is an inherent limitation of the xenogeneic NSG mouse model used.

In an earlier clinical study, retargeted T cells were generated fortherapy by loading pre-activated T cells with a bispecific mouse mAbOC/TR, directed to the CD3 molecule on T lymphocytes and to FRα on EOCcells (Canevari et al., 1988, Int J Cancer Suppl 2:18-21).Administration of FRα-redirected T cells to women with minimal residualovarian cancer resulted in antitumor responses in 27% of patients withmild to moderate immunotherapy-related toxicities; however, therapy waslimited by the inability to generate stable anti-FRα-specific T-cellmemory and the induction of human anti-mouse antibodies against thebispecific mAb in approximately 90% of treated patients (Canevari etal., 1995, J Natl Cancer Inst 87:1463-9). In a phase I study of anti-FRαCAR therapy for cancer, Kershaw and colleagues (Kershaw et al., 2006,Clin Cancer Res 12: 6106-15) transferred T cells that were retargeted toFRα by a first-generation MOv18 scFv-based CAR to immunocompetentpatients with advanced ovarian cancer. The parental MOv18 antibody has asimilar affinity for FRα (10′-109 M⁻¹) as MOv19 used in the present CARconstruct (Miotti et al., 1987, Int J Cancer 39:297-303; Figini et al.,1998, Cancer Res 58:991-6) though the relative affinities of their scFvproducts in CARs is not known. MOv18 and MOv19 also bindnon-cross-reactive epitopes (Miotti et al., 1987, Int J Cancer39:297-303), which may influence their relative ability to accesssurface antigen. Therapy using MOv18-ζ CAR was safe and feasible;however, no patient experienced a tumor response which was attributed toa lack of transferred T-cells persistence after infusion, poor tumorlocalization, and the development of a serum inhibitory factor thatreduced CAR T-cell activity in in vivo study (Kershaw et al., 2006, ClinCancer Res 12: 6106-15). Studies presented herein address these issues.Similar to the study of Kershaw and colleagues (Kershaw et al., 2006,Clin Cancer Res 12: 6106-15), first-generation MOv19-ζ CAR, whichredirected T-cell cytotoxicity in vitro, only delayed tumor progressionin vivo and CARs did not persist long-term in vivo. It is shown hereinthat tumor response and T-cell persistence can be evoked by provision ofCD137 costimulatory signals to anti-FRα CAR T cells, which isfacilitated principally by engagement of their CAR with tumor antigen.Moreover, transfer of MOv19-BBζ T cells leads to increased accumulationof human T cells in regressing ovarian cancer lesions. Although themouse anti-human MOv19 scFv used in the construction of the MOv19-BBζCAR is likely to elicit anti-mouse humoral responses in immunocompetentrecipients, as seen in past CAR studies and trials using MOv18 scFv(Kershaw et al., 2006, Clin Cancer Res 12: 6106-15; Canevari et al.,1995, J Natl Cancer Inst 87:1463-9; Lamers et al., 2011, Blood117:72-82), nonmyeloablative immunosuppressive preconditioning candisable host endogenous immunity to promote the in vivo persistence of Tcells expressing CARs and TCRs of mouse origin, facilitating tumorregression (Kochenderfer et al., 2010, Blood 116:4099-102; Berger etal., 2001, J Virol 75:799-808; Johnson et al., 2009, Blood 114:535-46).The use of immunodeficient NSG mice models T-cell transfer in thesetting of host lymphodepletion, albeit in the absence of humanderivatives and endogenous immune reconstitution. Based on the resultspresented herein, the use of fully human anti-FRα scFv candidates forthe next generation of CAR-redirected therapy is worthy of investigation(Figini et al., 1998, Cancer Res 58:991-6; Figini et al., 2009, CancerImmunol Immunother 58:531-46). Results presented herein support thenotion that incorporation of the CD137 signaling domain in FRα-specificCARs overcomes the limitations of past CAR approaches by improving thepersistence of transferred T cells in vivo, thereby increasing theirretention in tumor and bolstering antitumor potency. Carefulconsiderations must be made when targeting of self/tumor antigens withCARs or exogenous TCRs, which hold the potential for mediating seriousadverse events (Johnson et al., 2009, Blood 114:535-46; Morgan et al.,2010, Mol Ther 18:843-51); however, FRα, which is present on normaltissues, is localized primarily to the apical surfaces of polarizedepithelia, where it may be inaccessible to parenterally administeredfolate conjugates and redirected T cells (Low et al., 2004, Adv DrugDeliv Rev 56:1055-8).

Example 20: A Pilot Phase I Dose Escalation Study to Establish theSafety and Proof of Concept of Autologous Folate Receptor (α-FR)-AlphaRedirected T Cells Administration Intravenously in Patients withRecurrent Ovarian Cancer

The major question in the development of CAR T cells for cancer therapyis identifying vector designs that enhance the persistence of the cellspost infusion, and to optimize trafficking of CAR T cells to tumorsites. This study design tests the hypothesis that the changes in vectordesign improves the survival of CAR T cells in comparison to a previousstudy of ovarian cancer (Hwu et al., 2006, Clinical Cancer Research,12(20): 6106-6115). Described herein is a phase I study to determine thesafety, tolerability and feasibility of administering (CAR) T cellstransduced with the anti-α-FR Chimeric Antigen Receptor (CAR) insubjects with ovarian cancer receiving anti-α-FR CAR T cells. Theprotocol schema is shown in FIG. 28. At entry subjects are screened andtheir eligibility is determined. Those who meet all eligibility criteriaundergo apheresis within 4-6 weeks of screening to obtain peripheralblood mononuclear cells (PBMCs) for CAR T cell manufacturing. The Tcells are purified from the PBMC, transduced with anti-α-FR scFvexpanded in vitro and then frozen for future administration.

Investigational Agent and Dose

The study drug is autologous T cells that have been engineered toexpress a Chimeric Antigen Receptors (CAR) comprised of an extracellularsingle chain antibody (scFv) with specificity for α-FR and anintracellular TCRz chain and 4—-1BB signaling domain. The CAR constructswere developed, and the clinical grade pELNS lentiviral vector carryingthe MOv19-BBζ CAR has been already manufactured. The CAR T cells arecryopreserved in infusible cryomedia, and are administered in either 1or 2 bags. Each bag contains an aliquot (volume dependent upon dose) ofcryomedia containing the following infusible grade reagents (% v/v):31.25 μlasmalyte-A, 31.25 dextrose (5%), 0.45 NaCl, up to 7.50 DMSO,1.00 dextran 40, 5.00 human serum albumin with the appropriate number ofautologous T cells per bag.

Three T cell dose levels are tested, starting at the expected “MinimalAnticipated Biological Effect Level” (MABEL) dose of ˜3×10⁷ CAR+ Tcells/m² to reach an expected “No Observable Adverse Effect Level”(NOAEL) dose of ˜3×10⁸ CAR+ T cells/m².For additional safety, a “splitdose” approach to dosing is followed over 3 days, administeringCAR-transduced T cells by intravenous infusion using 10% of the totalintended on day 0, 30% on day 1 and 60% on day 2.

Patients receive a single dose of CAR T cells intravenously using a“split dose” regimen on day 0, 1 and 2 by rapid i.v. infusion. Theinfusion is scheduled to occur 2 days following chemotherapy.

Cohort 1 3×10⁷ CAR T cells/m² (with a minimally accepted dose of 2.5×10⁷and a maximally accepted dose of 3.5×10⁷)

Cohort 2 1×10⁸ CAR T cells/m² (with a minimally accepted dose of 8×10⁷and a maximally accepted dose of 1.2×10′)

Cohort 3 3×10⁸ CAR T cells/m² (with a minimally accepted dose of 2.6×10⁸and a maximally accepted dose of 3.4×10′)

Preparation

The CAR T cells are prepared in a production facility and are notreleased from the production facility until release criteria for theinfused cells (e.g., cell purity, sterility, average copy number ofvectors/cell, etc.) are met. Upon release, the cells are taken to aclinic. Bags (50 to 100 ml capacity) containing CAR-transduced T cellsare stored in blood bank conditions in a monitored −150° C. freezer atthe University of Pennsylvania. Infusion bags are stored in the freezeruntil needed.

Cell Thawing

Transduced T cells are transported on dry ice from the productionfacility a stem cell unit at the clinic where the product is released.The transduced T cells are transported by the research nurse/coordinatorto the patient's bedside in the clinic. The infusion takes place in anisolated room in the clinic. The cells are thawed at the bedside one bagat a time using a water bath maintained at 36° C. to 38° C. The bag isgently massaged until the cells have just thawed. It is made sure thatthere are no frozen clumps left in the container. If the CAR T cellproduct appears to have a damaged or leaking bag, or otherwise appearsto be compromised, it is not infused, and is returned to the productionfacility.

Return or Destruction of Study Drug

CAR T cells may require return to the production facility for a varietyof reasons, including but not limited to: 1) Mislabeled product; 2)Condition of patient prohibits infusion/injection, and 3) Subjectrefuses infusion/injection; any unused product are returned toproduction facility for disposal

Premedication

Side effects following T cell infusions include transient fever, chills,fatigue and/or nausea. It is recommended that the subjects bepre-medicated with acetaminophen 650 mg by mouth and diphenhydraminehydrochloride 25-50 mg by mouth or IV, prior to the infusion of CAR Tcells. These medications may be repeated every six hours as needed. Acourse of non-steroidal anti-inflammatory medication may be prescribedif the patient continues to have fever not relieved by acetaminophen. Itis recommended that patients not receive systemic corticosteroids suchas hydrocortisone, prednisone, prednisolone (Solu-Medrol) ordexamethasone (Decadron) at any time, except in the case of alife-threatening emergency, since this may have an adverse effect on Tcells. If corticosteroids are required for an acute infusional reaction,an initial dose of hydrocortisone 100 mg is recommended.

Administration of Study Drug

Cells are infused within approximately 10-40 minutes after thaw. Thetransduced T cells are administered on 3 consecutive days by rapidintravenous infusion at a flow rate of approximately 10 mL to 20 ml perminute through an 18-gauge latex free Y-type blood set with a 3-waystopcock. Dosing takes place by gravity infusion. If the infusion rateby gravity is too slow, the transduced T cell drug product is drawn intoa 50 mL syringe via the stopcock and manually infused at the requiredrate. The duration of the infusion is approximately 15 minutes. One ortwo bags of CAR T cells are delivered to the bedside on ice, and thecells are administered to the subject while cold. Each infusion bag hasaffixed to it a label containing the following: “FOR AUTOLOGOUS USEONLY.” In addition the label has at least two unique identifiers such asthe subject's initials, birth date, and study number. Prior to theinfusion, two individuals independently verify all this information inthe presence of the subject and so confirm that the information iscorrectly matched to the participant.

Emergency medical equipment (i.e., emergency trolley) is availableduring the infusion in case the subject has an allergic response, orsevere hypotensive crisis, or any other reaction to the infusion. Vitalsigns (temperature, respiration rate, pulse, and blood pressure) aretaken before and after infusion, then every 15 minutes for at least twohour and until these signs are satisfactory and stable. The subject isasked not to leave until the physician considers it is safe for him orher to do so.

Within 15 minutes (±5 minutes) following completion of dosing withtransduced T cells, a blood sample is obtained for a baselinedetermination of the number of transduced T cells.

Screening and Baseline Evaluation:

Patients sign the informed consent before testing begins. Screeningprocedures are done within 4-6 weeks of apheresis, and include:

-   -   A review of inclusion/exclusion criteria    -   Confirm an ECOG performance status <2    -   Tumor Burden Evaluation: performed as standard of care to        include CT scan chest, abdomen and pelvis. Does not have to be        repeated if done within 4 weeks prior to visit    -   Physical examination (including vital signs, height and weight,        medical and medication history)    -   Review of concomitant medications    -   Hematology: Complete blood count (CBC), differential, platelets,        Prothrombin Time (PT) and Partial Thromboplastin Time (PTT)    -   Serum Chemistries: BUN, creatinine, electrolytes, and glucose;        calcium, magnesium, phosphate, SGOT, SGPT, alkaline phosphatase,        LDH, total bilirubin, uric acid, total protein and albumin    -   Virology (screening): HIV-1, 2, HTLV-1/2, Hepatitis B (HbsAg,        α-HBc), Hepatitis C (αHCV).    -   Serum CA-125    -   Urinalysis    -   EKG (up to 6 weeks old, can be done outside of institution)    -   VSV-G antibody response and human anti-murine antibody (HAMA).    -   CT/MRI (up to 6 weeks old, can be done outside of institution)    -   Research Blood draws

Apheresis

A ˜10-15 liter apheresis procedure is carried out at the apheresiscenter. PBMC are obtained for CAR T cells during this procedure. From asingle leukapheresis, the intention is to harvest at least 50×10⁹ whiteblood cells to manufacture CAR T cells. Baseline blood leukocytes forFDA look-back requirements and for research are also obtained andcryopreserved. Without being held to any particular theory, the cellproduct is expected to be ready for release approximately 4 weeks later.A repeat apheresis may be offered during the course of the study if thetarget number of T cells was not reached.

Transient Lymphodepletion Regimen (Day −5 though −3)

Subjects receive a single course of outpatient conditioninglymphodepletion chemotherapy with intravenous cyclophosphamide (300mg/m²/d for 3 days) and intravenous fludarabine (30 mg/m²/d for 3 days)on Day −5 through Day −3. This is a well-tolerated outpatient regimen.Dose reduction to cyclophosphamide 250 mg/m²/d and fludarabine 25mg/m²/d is allowed at the discretion of the treating physician.

The following comprises a course of therapy for Day −5 through Day −3:

Subjects are pre-medicated with acetaminophen (Tylenol) 650 mg andhydrated with 0.9% Sodium Chloride with 10 meq/1 KCL at 2.6 ml/kg/hr(hydration is at the discretion of the Investigator).

Subjects receive daily Cyclophosphamide 300 mg/m²/d IV in 250 ml D5Wover 1 hr for 3 days. Maximum dose not to exceed doses calculated onbody weights greater than 140% of the maximum ideal body weight.

Subjects receive daily Fludarabine 30 mg/m²/day IVPB daily over 15-30minutes for 3 days. Maximum dose not to exceed doses calculated on bodyweights greater than 140% of the maximum ideal body weight (MetropolitanLife Insurance Company). The fludarabine is started approximately 1-2hours after the cyclophosphamide.

Antibiotics, Anti-fungals and Anti-virals are given to subjects asprophylaxis: The typical phrophylactic doses are: Altrex 500 mg daily,Bactrium DS one tablet q M W F and Fluconazole 200 mg daily. Theduration of medication is until Absolute Lymphocyte count (ALC) andAbsolute Neutrophil Count (ANC) count returns to pre medicationbaseline.

Patients are encouraged oral intake of fluids of 2-3 liters/day on theday prior to, during and following chemotherapy. Hematopoietic growthfactors are given as clinically indicated.

CAR T Cell Administration for First Treatment Cycle (Day 0, 1 & 2)

Subjects receive infusions in an isolated room. The cells are thawed atthe patient's bedside as described elsewhere herein. The thawed cellsare given at an infusion rate as quickly as tolerated so that theduration of each infusion is approximately 10-15 minutes. In order tofacilitate mixing, the cells are administered simultaneously using aY-adapter. A blood sample for determination of baseline CAR T cell levelis obtained before infusion and 20 minutes post infusion. Subjects areinfused and premedicated as described elsewhere herein. Subjects areobserved for at least 2 hours post infusion, with vital signs(temperature, respiration rate, pulse and blood pressure) monitoredevery 15 minutes for at least two hours and until these signs aresatisfactory and stable. Pulse oximetry determination of bloodoxygenation is used as means of pulmonary assessment prior to and 15minutes post T cell infusion and every 15 min thereafter until thecompletion of the observation period.

Subject Assessments

Subjects have the following done on Day 0, 1&2 before T cell infusion:

-   -   ECOG Performance Status    -   Physical Exam (including vital signs, weight, ConMed and Adverse        event assessment).    -   Hematology: CBC, differential and platelets, Prothrombin Time        (PT) and Partial Thromboplastin Time (PTT).    -   Serum Chemistries: BUN, creatinine, electrolytes, glucose,        calcium, SGOT, SGPT, alkaline phosphatase, total bilirubin,        total protein, albumin.    -   Urinalysis: random urine protein, random urine creatinine to        measure urine protein: creatinine (UPC) ratio. 24-hour urine        protein is determined in subjects with proteinuria greater than        +1 in the absence of UTI.    -   Serum CA-125    -   Serum HAMA and VSV-G level    -   Research blood draws

Subjects have the following done on Day 7, 14, 25 and 39 post T cellinfusion:

-   -   ECOG Performance Status    -   Physical Exam (including vital signs, weight, ConMed and Adverse        event assessment).    -   Hematology: CBC, differential and platelets, Prothrombin Time        (PT) and Partial Thromboplastin Time (PTT).    -   Serum Chemistries: BUN, creatinine, electrolytes, glucose,        calcium, SGOT, SGPT, alkaline phosphatase, total bilirubin,        total protein, albumin.    -   Urinalysis: random urine protein, random urine creatinine to        measure urine protein: creatinine (UPC) ratio. 24-hour urine        protein is determined in subjects with proteinuria greater than        +1 in the absence of UTI.    -   EKG    -   Serum HAMA and VSV-G level    -   Research blood draws

Subjects undergo a CT guided tumor biopsy around Day 39.

-   -   Pre and Post Infusion Laboratories to Assess Safety and        Engraftment

Subjects are asked to undergo ˜100 ml phlebotomy (2 red tops and 3 greentops) to evaluate the presence and safety of CAR T cells and forcollection of immunological data on the following time points during thefirst treatment cycle: Day −5 (prior to lymphodepletion), Day 0 (priorto T cell infusion), 15 minutes and 2 hours after each T cell infusion,then daily till Day 7, then again on Day 9, 11, 14, 18, 25 then onceevery 2 weeks until EOS.

At EOS (Day 58), an additional of ˜200 ml phlebotomy (5 green tops and 3red tops) is collected. All subjects undergo ˜6 ml phlebotomy on firstand third day of cyclophosphamide chemotherapy prior to chemotherapyinfusion; and prior to T cell infusion; twice weekly thereafter till ANCand ALC reach pretreatment baseline or 1500 and 1000 respectively SerumCA-125 levels are recorded at least monthly during the study, but arenot be included in clinical decision-making.

End of Study Evaluations (EOS, Day ˜58)

-   -   Specific monitoring tests and procedures are completed on Day        ˜58 as follows:    -   ECOG Performance Status    -   Physical Exam (including vital signs, weight, ConMed and Adverse        event assessment).    -   Hematology: CBC, differential and platelets, Prothrombin Time        (PT) and Partial Thromboplastin Time (PTT).    -   Serum Chemistries: BUN, creatinine, electrolytes, glucose,        calcium, SGOT, SGPT, alkaline phosphatase, total bilirubin,        total protein, albumin, LDH, magnesium, phosphate, uric acid.    -   Serum CA-125    -   Tumor Burden Evaluation: CT/MRI scan of chest, abdomen and        pelvis    -   EKG    -   Subjects have ˜200 ml phlebotomy for immunologic monitoring (5        green tops and 2 red tops).C    -   CT/MRI

On Day 58 (End of Study (EOS)), subjects have completed the firsttreatment cycle and have undergone immune and clinical assessment (asmeasured by immune-related response criteria). Subjects who achieveimmune related Complete Response (ir-CR) have the option to receiveanother cycle upon progression of disease. Those who experienceir-Partial Response (ir-PR), ir-Stable Disease (ir-SD) or ir-progressionof disease (ir-PD) have the option to receive another treatment cycleafter lymphodepletion. Subjects only receive more than one cycle if theall safety parameters are met and it is safe to move to the next cycle.Subject has to also have genetically engineered T cells available.

Primary Endpoints

Primary endpoints of the study include:

-   -   Safety: Monitor the occurrence of study related adverse events        (defined as ≥Grade 3 signs/symptoms, laboratory toxicities, and        clinical events, with some exceptions noted previously) that are        “possibly”, “likely”, or “definitely” related to study treatment        any time from the first day of study treatment until EOS.    -   Feasibility: Feasibility is defined as the number of        manufactured products that do not meet release criteria for        vector transduction efficiency. T cell purity, viability, and        sterility is determined (defined as

“manufacturing failures”).

Secondary Endpoints

The major secondary endpoints of the study include:

-   -   Persistence and Engraftment of CAR T cells: Engraftment of CAR T        cells is evaluated post dosing by DNA PCR for vector copy number        in PBMC.

The number of anti-α-folate receptor CAR T cells in the blood ismeasured by RT-PCR performed ˜15 minutes, 2 hours after each T cellinfusion then daily till Day 7, then again on Day 9, 11, 14, 18, 25 thenonce every two weeks till end of study. The Optimal Biologic Dose (OBD)is defined by comparing the dose levels for safety profile andengraftment of CAR T cells in circulation and tumor biopsies; the OBDhas the highest engraftment at day 28 with an acceptable toxicityprofile.

-   -   Clinical Efficacy: Immune related response, the distribution of        progression-free survival, overall survival and time to        progression for patients treated with CAR T cells following        lymphodepletion with cyclophosphamide/fludarabine is determined.    -   The effect of CAR T cells on tumor immunity and α-folate        receptor expression is determined using research laboratory        assays.    -   Persistence, Engraftment, Phenotype and Function of CAR+ T        cells: FRα CAR+ T cells are readily identified by flow cytometry        using PE conjugated goat anti-mouse IgG F(ab′)2 (Jackson        ImmunoResearch). CAR+ T cells are quantified in peripheral blood        longitudinally (˜15 minutes, 24 hrs, 48 hrs, 72 hrs, Day, 7, 14,        21, and 28 days as well as at 6, 8, 12, 16 weeks and every 6        months after dosing. In addition, CAR+ T cells are detected by        DNA quantitative (q)PCR for vector copy number in PBMC, an        acquisitively sensitive method to test for persistence of CAR+ T        cells. Phenotypic analysis of CAR+ T cells includes detailed        interrogation for memory cell (CCR7, CD62L, CD45RA, CD27, CD28,        Fas etc) vs. effector cell markers (CD45R0, CCR6, CD25, CD38,        HLADR, GITR, PD1 etc). CAR+ T cells are also phenotyped for IL-7        receptor CD127 and IL-15 receptor alpha expression. Ex vivo        stimulation with PHA-ionomycin or cognate antigen followed by        interrogation of intracellular cytokines (INFγ, TNFα, IL-2,        IL-17, IL-4, TGFβ, IL-10), granzymes, CD137 and CD107a provides        a detailed and longitudinal characterization of in vivo        polarization and function post transfer. The presence of CAR+ T        cells is quantified in tumor biopsies by DNA qPCR and correlated        with FRα protein expression at baseline and end of study.    -   CAR immunogenicity: The development of host immune responses to        the CAR T cells by HAMA and VSV-G ELISA is assessed and        correlated with engraftment of CAR+ T cells.    -   Effect of CAR+ T cells on tumor microenvironment: Detailed        leukocyte subset infiltrate analysis are performed by        immunohistochemistry, and comprehensive immune analysis of the        tumor microenvironment is done by multiplex qPCR and/or        Affymetrix arrays.    -   Dose optimization: The OBD is defined by comparing the dose        levels for safety profile and engraftment of CAR+ T cells in        circulation and tumor biopsies; the OBD has the highest        engraftment at day 28 with an acceptable toxicity profile.

Number of modified T-cells in serum, HAMA levels, serum ELISPOT measuresof host immunity to anti-α-folate receptor and immune function, as wellas VSV-G antibody response are displayed graphically as a function oftime. The mean levels of α-folate receptor expression between tumorswith and without intratumoral anti-α-folate transduced cells arecomputed for those patients who receive tissue biopsy. 95% confidenceintervals for proportions and means are computed.

Cytokine measurements are conducted using Luminex and evaluating a panelof cytokines/chemokines/immune factors with potential to be modulated bythe treatment. The panel is composed of all or a subset of the followingfactors: IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF.These measurements are conducted on serum samples collected on Day −5(prior to lymphodepletion), Day 0 (prior to T cell infusion), 15 minutesand 2 hours after each T cell infusion, then daily till Day 7, thenagain on Day 9, 11, 14, 18, 25 then once every 2 weeks until EOS. Thishelps to determine IL-7's profile in the first three cohorts and helpbetter determine rhIL-7's administration schedule in a future cohort.

Clinical Efficacy

Anti-tumor activity is reported as a secondary trial endpoint. Thepurpose of this trial is to determine early on in clinical developmentof CAR T cells the persistence and engraftment of these cells using theIV route of administration. Response and progression is evaluated inthis study using the new immune-related response criteria.

Definition of Tumor Response Using irRC

The sum of the products of diameters at tumor assessment using theimmune-related response criteria (irRC) for progressive diseaseincorporates the contribution of new measurable lesions. Each netPercentage Change in Tumor Burden per assessment using irRC criteriaaccounts for the size and growth kinetics of both old and new lesions asthey appear (Wolchok et al., 2009, Clinical Cancer Research, 15(23):7412-7420; Hoos et al., 2010, Journal of the National Cancer Institute,102(18): 1388-1397; Hodi, 2010, New England Journal of Medicine,363(13): 1290)

Definition of Index Lesions Response Using irRC

-   -   irComplete Response (irCR): Complete disappearance of all index        lesions.    -   irPartial Response (irPR): Decrease, relative to baseline, of        50% or greater in the sum of the products of the two largest        perpendicular diameters of all index and all new measurable        lesions (ie., Percentage Change in Tumor Burden). Note: the        appearance of new measurable lesions is factored into the        overall tumor burden, but does not automatically qualify as        progressive disease until the SPD increases by ≥25% when        compared to SPD at nadir.    -   irStable Disease (irSD). Does not meet criteria for irCR or        irPR, in the absence of progressive disease.    -   irProgressive Disease (irPD): At least 25% increase Percentage        Change in Tumor Burden (i.e., taking sum of the products of all        index lesions and any new lesions) when compared to SPD at        nadir.

Definition of Non-Index Lesions Response Using irRC

-   -   irComplete Response (irCR): Complete disappearance of all        non-index lesions.    -   irPartial Response (irPR) or irStable Disease (irSD): non-index        lesion(s) are not considered in the definition of PR, these        terms do not apply.    -   irProgressive Disease (irPD): Increases in number or size of        non-index lesion(s) does not constitute progressive disease        unless/until the Percentage Change in Tumor Burden increases by        25% (i.e., the SPD at nadir of the index lesions increases by        the required amount).

Impact of New Lesions on irRC

New lesions in and by themselves do not qualify as progressive disease.However their contribution to total tumor burden is included in the SPD,which in turn feeds into the irRC criteria for tumor response.Therefore, new non-measurable lesions do not discontinue any subjectfrom the study.

Definition of Overall Response Using irRC

Overall response using irRC is based on these criteria:

-   -   Immune-Related Complete Response (irCR): Complete disappearance        of all tumor lesions (index and nonindex together with no new        measurable/unmeasurable lesions) for at least 4 weeks from the        date of documentation of complete response.    -   Immune-Related Partial Response (irPR): The sum of the products        of the two largest perpendicular diameters of all index lesions        is measured and captured as the SPD baseline. At each subsequent        tumor assessment, the sum of the products of the two largest        perpendicular diameters of all index lesions and of new        measurable lesions are added together to provide the Immune        Response Sum of Product Diameters (irSPD). A decrease, relative        to baseline of the irSPD compared to the previous SPD baseline,        of 50% or greater is considered an immune Partial Response        (irPR).    -   Immune-Related Stable Disease (irSD): irSD is defined as the        failure to meet criteria for immune complete response or immune        partial response, in the absence of progressive disease.    -   Immune-Related Progressive Disease (irPD): It is recommended in        difficult cases to confirm PD by serial imaging. Any of the        following constitutes progressive disease:        -   At least 25% increase in the sum of the products of all            index lesions over baseline SPD calculated for the index            lesions.        -   At least a 25% increase in the sum of the products of all            index lesions and new measurable lesions (irSPD) over the            baseline SPD calculated for the index lesions.

Yearly Evaluations 1 to 15 Years Post Infusion

At the end of the study, patients have long-term follow up for up to 15years in accordance with recent guidelines for long term follow-up(LTFU) set forth by the ASGT and the FDA. LTFU requires 6 months visitsfor the first 5 years post infusion, and then annual visits if thevector modified cells are no longer detected in the blood. Visitsinvolve blood draws and a physical exam.

Data Collection and Follow-up for Withdrawn Subjects

Follow-up data collection after cell therapy clinical trials forsubjects who receive the study drug is up to 15 years in accordance withFDA guidelines. As long as patients have detectable cells transducedwith the scFv chimeric receptor, they are followed for toxicity, immunereactions, and any long-term adverse events. Many patients who respondto cell therapy may also have prolonged DFS but are also at risk forlate relapse. The intent is to follow all patients treated with CAR Tcells indefinitely at least until the time alternative treatment isrequired for their disease, and/or they are no longer at risk fortoxicity from the infused cells (i.e. loss of engraftment). Therefore,data collection is continued regarding 1) engraftment as long aspatients are at risk (until evidence of loss of detectable transduced Tcells); 2) DFS until there is disease progression; 3) survival until thetime of death or 4) until the patient withdraws consent for clinicaldata collection.

Patients who are followed at other institutions or practices, because ofpreference or geographical concerns have follow-up via notes from theirlocal physician and/or phone interviews with periodic study assessments.An example would be a patient referred from out of state but cared forat another center. Toxicity and other clinical assessments are obtainedfrom the treating physician. Every effort is made to contact subjectswho appear to be lost to follow-up in order to at least obtain survivaldata. In the event a subject fails to complete the follow-uprequirements, documentation of all attempts to contact the subjectincludes at least 3 telephone contacts (on different days and atdifferent times of the day), and a certified letter. Subjects arewithdrawn from DFS assessments if 1) there is evidence for lack ofresponse, relapse or progressive disease after 6 months of follow-up or2) at any time they require new treatment for their disease (i.e.conventional chemotherapy). Subjects are withdrawn from survivalassessments at the time of death.

Example 21: Humanized Anti-FR CAR Recognize and Respond to FR ExpressingCancer Cell Lines

A fully-human anti-FR CAR was constructed comprising the humanized C4scFV. Primary human T cells were transduced to express the humanizedanti-FR CAR, and the humanized anti-FR CAR was efficiently expressed onthe surface of transduced T cells (FIGS. 29A-29B). Transduced anduntransduced T cells were co-cultured overnight with ovarian or breastcancer cells. Humanized anti-FR CAR transduced T cells recognized FRexpressing cell lines in vitro, as transduced, but not untransduced, Tcells secreted IFN-γ when co-cultured with FR expressing cell lines.Cell lines that expressed little or no FR (A2780 and C30) were notrecognized by transduced T cells (FIGS. 29A-29B).

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. An isolated nucleic acid sequence encoding a chimeric antigenreceptor (CAR), wherein the isolated nucleic acid sequence comprises anucleic acid sequence encoding an α-folate receptor (FRα) antibodycomprising the amino acid sequence of SEQ ID NO:
 15. 2. The isolatednucleic acid sequence of claim 1, wherein the CAR further comprises aCD3 zeta signaling domain.
 3. The isolated nucleic acid sequence ofclaim 2, wherein the CD3 zeta signaling domain comprises an amino acidsequence of SEQ ID NO:
 19. 4. The isolated nucleic acid sequence ofclaim 2, wherein the CD3 zeta signaling domain is encoded by a nucleicacid sequence of SEQ ID NO:
 7. 5. The isolated nucleic acid sequence ofclaim 1, wherein the CAR further comprises a transmembrane domain.
 6. Anisolated chimeric antigen receptor (CAR), wherein the CAR comprises anα-folate receptor (FRα) antibody comprising the amino acid sequence ofSEQ ID NO:
 15. 7. The isolated CAR of claim 6, wherein the CAR furthercomprises a CD3 zeta signaling domain.
 8. The isolated CAR of claim 7,wherein the CD3 zeta signaling domain comprises an amino acid sequenceof SEQ ID NO:
 19. 9. The isolated CAR of claim 7, wherein the CD3 zetasignaling domain is encoded by a nucleic acid sequence of SEQ ID NO: 7.10. The isolated CAR of claim 6, wherein the CAR further comprises atransmembrane domain.
 11. A method for providing anti-tumor immunity ina subject, the method comprising: administering to the subject aneffective amount of a T cell comprising the CAR of claim 6, therebyproviding anti-tumor immunity in the subject.
 12. The method of claim11, wherein the CAR further comprises a 4-1BB costimulatory domain 13.The method of claim 12, wherein the 4-1BB costimulatory domain comprisesan amino acid sequence of SEQ ID NO:
 18. 14. The method of claim 12,wherein the 4-1BB costimulatory domain is encoded by the nucleic acidsequence of SEQ ID NO:
 6. 15. A method for stimulating a T cell-mediatedimmune response to a cell population or tissue in a subject, the methodcomprising: administering to the subject an effective amount of a T cellcomprising the CAR of claim 6, thereby stimulating a T cell-mediatedimmune response in the subject.
 16. The method of claim 15, wherein theCAR further comprises a 4-1BB costimulatory domain.
 17. The method ofclaim 16, wherein the 4-1BB costimulatory domain comprises an amino acidsequence of SEQ ID NO:
 18. 18. The method of claim 16, wherein the 4-1BBcostimulatory domain is encoded by the nucleic acid sequence of SEQ IDNO:
 6. 19. A method for treating an ovarian cancer in a subject, themethod comprising: administering to the subject an effective amount of aT cell comprising the CAR of claim 6, thereby treating the ovariancancer in the subject.
 20. The method of claim 19, wherein the CARfurther comprises a 4-1BB costimulatory domain
 21. The method of claim20, wherein the 4-1BB costimulatory domain comprises an amino acidsequence of SEQ ID NO:
 18. 22. The method of claim 20, wherein the 4-1BBcostimulatory domain is encoded by the nucleic acid sequence of SEQ IDNO:
 6. 23. A method for treating cancer in a subject, the methodcomprising: administering to the subject an effective amount of a T cellcomprising the CAR of claim 6, thereby treating cancer in the subject.24. The method of claim 23, wherein the CAR further comprises a 4-1BBcostimulatory domain
 25. The method of claim 24, wherein the 4-1BBcostimulatory domain comprises an amino acid sequence of SEQ ID NO: 18.26. The method of claim 24, wherein the 4-1BB costimulatory domain isencoded by the nucleic acid sequence of SEQ ID NO:
 6. 27. A method ofgenerating a persisting population of genetically engineered T cells ina subject diagnosed with ovarian cancer, the method comprising:administering to the subject an effective amount of a T cell comprisingthe CAR of claim 6, wherein the persisting population of geneticallyengineered T cells persists in the subject for at least one month afteradministration.
 28. The method of claim 27, wherein the CAR furthercomprises a 4-1BB costimulatory domain
 29. The method of claim 28,wherein the 4-1BB costimulatory domain comprises an amino acid sequenceof SEQ ID NO:
 18. 30. The method of claim 28, wherein the 4-1BBcostimulatory domain is encoded by the nucleic acid sequence of SEQ IDNO:
 6. 31. The isolated nucleic acid sequence of claim 1, wherein theCAR comprises the amino acid sequence of SEQ ID NO:
 13. 32. The isolatednucleic acid sequence of claim 1, wherein the CAR is encoded by thenucleic acid sequence of SEQ ID NO:
 1. 33. The isolated CAR of claim 6,wherein the CAR comprises the amino acid sequence of SEQ ID NO:
 13. 34.The isolated CAR of claim 6, wherein the CAR is encoded by the nucleicacid sequence of SEQ ID NO:
 1. 35. The method of claim 11, wherein theCAR comprises the amino acid sequence of SEQ ID NO:
 13. 36. The methodof claim 11, wherein the CAR is encoded by the nucleic acid sequence ofSEQ ID NO:
 1. 37. The method of claim 15, wherein the CAR comprises theamino acid sequence of SEQ ID NO:
 13. 38. The method of claim 15,wherein the CAR is encoded by the nucleic acid sequence of SEQ ID NO: 1.39. The method of claim 19, wherein the CAR comprises the amino acidsequence of SEQ ID NO:
 13. 40. The method of claim 19, wherein the CARis encoded by the nucleic acid sequence of SEQ ID NO:
 1. 41. The methodof claim 23, wherein the CAR comprises the amino acid sequence of SEQ IDNO:
 13. 42. The method of claim 23, wherein the CAR is encoded by thenucleic acid sequence of SEQ ID NO:
 1. 43. The method of claim 27,wherein the CAR comprises the amino acid sequence of SEQ ID NO:
 13. 44.The method of claim 27, wherein the CAR is encoded by the nucleic acidsequence of SEQ ID NO: 1.